Methods for arbitrary peptide synthesis

ABSTRACT

Methods, apparatus, systems, computer programs and computing devices related to biologically assembling and/or synthesizing peptides and/or proteins are disclosed.

RELATED APPLICATIONS

For purposes of the USPTO extra-statutory requirements, the presentapplication constitutes a continuation-in-part of U.S. patentapplication Ser. No. 11/478,540, entitled METHODS FOR ARBITRARY PEPTIDESYNTHESIS, naming Roderick A. Hyde; Edward K. Y. Jung and Lowell L.Wood, Jr. as inventors, filed 29, Jun. 2006.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and claims the benefit of theearliest available effective filing date(s) from the following listedapplication(s) (the “Related Applications”) (e.g., claims earliestavailable priority dates for other than provisional patent applicationsor claims benefits under 35 USC §119(e) for provisional patentapplications, for any and all parent, grandparent, great-grandparent,etc. applications of the Related Application(s)).

The United States Patent Office (USPTO) has published a notice to theeffect that the USPTO's computer programs require that patent applicantsreference both a serial number and indicate whether an application is acontinuation or continuation-in-part. Stephen G. Kunin, Benefit ofPrior-Filed Application, USPTO Official Gazette Mar. 18, 2003. Thepresent applicant entity has provided above a specific reference to theapplication(s) from which priority is being claimed as recited bystatute. Applicant entity understands that the statute is unambiguous inits specific reference language and does not require either a serialnumber or any characterization, such as “continuation” or“continuation-in-part,” for claiming priority to U.S. patentapplications. Notwithstanding the foregoing, applicant entityunderstands that the USPTO's computer programs have certain data entryrequirements, and hence applicant entity is designating the presentapplication as a continuation-in-part of its parent applications as setforth above, but expressly points out that such designations are not tobe construed in any way as any type of commentary and/or admission as towhether or not the present application contains any new matter inaddition to the matter of its parent application(s).

All subject matter of the Related Applications and of any and allparent, grandparent, great-grandparent, etc. applications of the RelatedApplications is incorporated herein by reference to the extent suchsubject matter is not inconsistent herewith.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of an illustrative apparatus in whichembodiments may be implemented.

FIG. 2 shows schematics of illustrative embodiments of the apparatus ofFIG. 1, with illustrative examples of a peptide synthesizer unit.

FIG. 3 shows schematics of illustrative embodiments of the apparatus ofFIG. 1, with specific examples of a sourcing unit.

FIG. 4 shows schematics of illustrative embodiments of the apparatus ofFIG. 1, with illustrative examples of a monitoring unit.

FIG. 5 shows schematics of illustrative embodiments of the apparatus ofFIG. 1, with illustrative examples of a controller unit.

FIG. 6 shows schematics of illustrative embodiments of the apparatus ofFIG. 1, with illustrative examples of a computing unit.

FIG. 7 shows an operational flow representing illustrative embodimentsof operations related to determining temporal-spatial parameters forsynthesizing one or more peptides based on a first possible dataset.

FIG. 8 shows optional embodiments of the operational flow of FIG. 7.

FIG. 9 shows optional embodiments of the operational flow of FIG. 7.

FIG. 10 shows optional embodiments of the operational flow of FIG. 7.

FIG. 11 shows optional embodiments of the operational flow of FIG. 7.

FIG. 12 shows optional embodiments of the operational flow of FIG. 7.

FIG. 13 shows a partial view of an illustrative embodiment of a computerprogram product that includes a computer program for executing acomputer process on a computing device.

FIG. 14 shows an illustrative embodiment of a system in whichembodiments may be implemented.

FIG. 15 shows an operational flow representing illustrative embodimentsof operations related to determining temporal-spatial parameters forco-localizing and/or separating one or more target components based on afirst possible dataset.

FIG. 16 shows optional embodiments of the operational flow of FIG. 15.

FIG. 17 shows optional embodiments of the operational flow of FIG. 15.

FIG. 18 shows optional embodiments of the operational flow of FIG. 15.

FIG. 19 shows optional embodiments of the operational flow of FIG. 15.

FIG. 20 shows optional embodiments of the operational flow of FIG. 15.

FIG. 21 shows optional embodiments of the operational flow of FIG. 15.

FIG. 22 shows a partial view of an illustrative embodiment of a computerprogram product that includes a computer program for executing acomputer process on a computing device.

FIG. 23 shows an illustrative embodiment of a system in whichembodiments may be implemented.

FIG. 24 shows a schematic of an illustrative apparatus for biologicallysynthesizing peptides.

FIG. 25 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa microchip as part of the reaction chamber.

FIG. 26 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa dispensensing unit.

FIG. 27 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa dispensing unit.

FIG. 28 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 29 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 30 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 31 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 32 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 33 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 34 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 35 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 36 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa dispensensing unit.

FIG. 37 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

FIG. 38 shows a schematic of an illustrative embodiment of theillustrative apparatus of FIG. 24, including an illustrative example ofa reaction chamber.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

This disclosure is drawn, inter alia, to methods, apparatus, computerprograms and computing devices related to biologically assembling and/orsynthesizing peptides and/or proteins.

As used herein, the term “peptide, peptides, protein, proteins” meanspolypeptide molecules formed from linking various amino acids in adefined order. The link between one amino acid residue and the nextforms a bond, including but not limited to an amide or peptide bond, orany other bond that can be used to join amino acids. Thepeptides/proteins may include any polypeptides of two or more amino acidresidues. The peptides/proteins may include any polypeptides including,but not limited to, ribosomal peptides and non-ribosomal peptides. Thepeptides/proteins may include natural and unnatural amino acid residues.The number of amino acid residues optionally includes, but is notlimited to, at least 5, 10, 25, 50, 100, 200, 500, 1,000, 2,000 or 5,000amino acid residues. The number of amino acid residues optionallyincludes, but is not limited to, 2 to 5,000, 2 to 2,000, 2 to 1,000, 2to 500, 2 to 250, 2 to 100, 2 to 50, 2 to 25, 2 to 10, 5 to 5,000, 5 to2,000, 5 to 1,000, 5 to 500, 5 to 250, 5 to 100, 5 to 50, 10 to 5,000,10 to 2,000, 10 to 1,000, 10 to 500, 10 to 250, 10 to 100, or 10 to 50.

As used herein, the term “amino acid or amino acids” means any moleculethat contains both amino and carboxylic acid functional groups,including, but not limited to, alpha amino acids in which the amino andcarboxylate functionalities are attached to the same carbon, theso-called α-carbon. Amino acids may include natural amino acids,unnatural amino acids, and arbitrary amino acids.

As used herein, the term “natural amino acid” includes, but is notlimited to, one or more of the amino acids encoded by the genetic code.The genetic codes of all known organisms encode the same 20 amino acidbuilding blocks with the rare exception of selenocysteine andpyrrolysine (Methods (2005) 36:227-238). In some embodiments, naturalamino acids may also include, but not be limited to, any one or more ofthe amino acids found in nature. In some embodiments, these naturalamino acids may include, but not be limited to, amino acids from one ormore of plants, microorganisms, prokaryotes, eukaryotes, protozoa orbacteria. In some embodiments, natural amino acids may include, but arenot limited to, amino acids from one or more of mammals, yeast,Escherichia coli, or humans.

As used herein, the term “unnatural amino acid” may include any aminoacid other than the amino acids encoded by the genetic code. In someembodiments, unnatural amino acids may include, but not be limited to,modified or derivatized amino acids encoded by the genetic code. In someembodiments, unnatural amino acids may include, but not be limited to,modified or derivatized amino acids of any one or more of the aminoacids found in nature. In some embodiments, unnatural amino acids mayinclude, but not be limited to, modified or derivatized amino acids fromone or more of plants, microorganisms, prokaryotes, eukaryotes,protozoa, bacteria, mammals, yeast, E. coli, or humans.

Unnatural amino acids are known in the art including, but not limitedto, those containing spectroscopic probes, post-translationalmodification, metal chelators, photoaffinity labels, D-enantiomers, aswell as other functional groups and modified structures (e.g. Methods 36(2005) 227-238; Annu. Rev. Biochem. (2004) 73:147-176; Science (2003)301:964-967; Royal Society of Chemistry (2004) 33:422-430).

As used herein, the term “arbitrary amino acid or arbitrary amino acids”means any amino acid that is not the amino acid coded for by the tRNAcodon recognition site as determined by the genetic code. In someembodiments, arbitrary amino acids are natural or unnatural amino acids.

As used herein, the term “amino acid residue or amino acid residues”means the remainder of an amino acid incorporated into apeptide/protein.

As used herein, the term “tRNA, tRNAs, transfer RNA or transfer RNAs”means an RNA chain that transfers an amino acid to a growing polypeptidechain. The tRNA has sites for amino acid attachment and codonrecognition. In some embodiments, tRNA includes natural, unnatural, andarbitrary tRNA.

As used herein, the term “natural tRNA” means one or more tRNA known innature that transfer an amino acid to a growing polypeptide chain. Insome embodiments, natural tRNA includes, but is not limited to, tRNAthat transfer one or more of the natural amino acids that are encoded bythe genetic code. In some embodiments, natural tRNA include, but are notlimited to, natural tRNA from one or more of plants, microorganisms,prokaryotes, eukaryotes, protozoa, bacteria, mammals, yeast, E. coli,humans, or archae.

As used herein, the term “unnatural tRNA” means any tRNA, other thantRNA known in nature, which transfers an amino acid to a growingpolypeptide chain. In some embodiments, unnatural tRNA may include, butare not limited to, modified or derivatized natural tRNA. In someembodiments, unnatural tRNA may include, but are not limited to,modified or derivatized natural tRNA from one or more of plants,microorganisms, prokaryotes, eukaryotes, protozoa, bacteria, mammals,yeast, E. coli, humans, or archae. In some embodiments, unnatural tRNAmay include, but are not limited to, tRNA with altered sites for aminoacid attachment, and/or tRNA with altered acceptor stems, and/or tRNAwith altered sites for codon recognition (the anticodon). In someembodiments, unnatural tRNA is recombinant tRNA.

As used herein, the term “arbitrary tRNA” means a tRNA that has beenmodified or derivatized such that the amino acid attachment site maybind one or more amino acids other than the amino acid specified by thecodon recognition site based on the genetic code. The amino acid may benatural or unnatural. Arbitrary tRNA may also include tRNA that havebeen modified or derivatized such that the amino acid attachment sitemay bind one or more different amino acids (natural or unnatural), whilethe codon recognition site may recognize one or more of one or more stopcodons, one or more singlet codons, one or more doublet codons, one ormore triplet codons, one or more quadruplet codons, one or morequintuplet codons, one or more sextuplet codons or others. Stop codonsinclude ochre (TAA), amber (TAG and opal (TGA).

Methods for modifying tRNA including, but not limited to, theanti-codon, the amino acid attachment site, and/or the accepter stem toallow incorporation of unnatural and/or arbitrary amino acids are knownin the art (Methods (2005) 36:227-238 Methods (2005) 36:270-278; Annu.Rev. Biochem. (2004) 73:147-176; Nucleic Acids Research (2004)32:6200-6211 PNAS (2003) 100:6353-6357; Royal Society of Chemistry(2004) 33:422-430).

As used herein, the term “anti-stop codon tRNA” means a tRNA having astop codon recognition site. In some embodiments, the anti-stop codontRNA may be charged with one or more natural, one or more unnatural, orone or more arbitrary amino acids. In some embodiments, the anti-stopcodon tRNA may be a natural, an unnatural, or an arbitrary tRNA.

As used herein, the term “charged tRNA or charged tRNAs” means tRNA thathas an amino acid bound at the amino acid attachment site. Duringpeptide synthesis, the aminoacyl group is transferred to the nascentpeptide, releasing the tRNA. As used herein, the term “released tRNA”means the tRNA remaining after the charged tRNA has donated the attachedamino acid to the nascent polypeptide. In some embodiments, the chargedtRNA may be natural, unnatural and/or arbitrary.

As used herein, the term “natural charged tRNA” means a natural tRNAthat has an amino acid bound at the amino acid attachment site. In someembodiments, the natural tRNA has one or more of a natural or anunnatural amino acid bound at the amino acid attachment site.

As used herein, the term “unnatural charged tRNA” means an unnaturaltRNA that has an amino acid bound at the amino acid attachment site. Insome embodiments, the unnatural tRNA has one or more of a natural or anunnatural amino acid bound at the amino acid attachment site.

As used herein, the term “arbitrary charged tRNA or tRNA charged witharbitrary amino acids” means a tRNA that has an amino acid bound at theamino acid attachment site, and that amino acid is different from theamino acid specified by the codon recognition site of the tRNA based onthe genetic code. In some embodiments, the bound amino acid is a naturalor an unnatural amino acid. In some embodiments, the codon recognitionsite includes, but is not limited to, a stop codon recognition site, asinglet codon recognition site, a doublet codon recognition site, atriplet codon recognition site, a quadruplet codon recognition site, aquintuplet codon recognition site, or a sextuplet codon recognitionsite.

As used herein, the term “charging or aminoacylation” is a process ofadding an aminoacyl group to a compound. Methods for charging natural,unnatural and/or arbitrary tRNA with natural, unnatural and/or arbitraryamino acids are known in the art, and include, but are not limited to,chemical aminoacylation, biological misacylation, acylation by modifiedaminoacyl tRNA synthetases, ribozyme-based, and protein nucleicacid-mediated methods (Methods (2005) 36:227-238; Methods (2005)36:39-244; Methods (2005) 36:245-251; Methods (2005) 36:270-278; Methods(2005) 36:291-298; Annu. Rev. Biochem. (2004) 73:147-176; Nucleic AcidsResearch (2004) 32:6200-6211; Royal Society of Chemistry (2004)33:422-430); Nature (2002) 20:723-728.

As used herein, the term “aminoacyl tRNA synthetase or aaRs” means anenzyme that catalyzes the binding of one or more amino acids to a tRNAto form an aminoacyl-tRNA (or charged tRNA). In some embodiments, thesynthetase binds the appropriate amino acid to one or more tRNAmolecules. In some embodiments, the synthetase mediates a proofreadingreaction to ensure high fidelity of tRNA charging. In some embodiments,the synthetase does not mediate a proofreading reaction to ensure highfidelity of tRNA charging.

As used herein, the term “natural aminoacyl tRNA synthetases” meansaminoacyl tRNA synthetases known in nature that add an aminoacyl groupto a tRNA. In some embodiments, natural aminoacyl tRNA synthetasesinclude, but are not limited to, aminoacyl tRNA synthetases that add oneor more of the natural aminoacyl groups that are encoded by the geneticcode. In some embodiments, natural aminoacyl tRNA synthetases include,but are not limited to, natural aminoacyl tRNA synthetases from one ormore of plants, microorganisms, prokaryotes, eukaryotes, protozoa,bacteria, mammals, yeast, Escherichia coli, or humans.

The term “unnatural aminoacyl tRNA synthetase” means any aminoacyl tRNAsynthetase, other than aminoacyl tRNA synthetases known in nature thatadd an aminoacyl group to a tRNA. In some embodiments, unnaturalaminoacyl tRNA synthetases may include, but are not limited to, modifiedor derivatized natural aminoacyl tRNA synthetases. In some embodiments,unnatural aminoacyl tRNA synthetases may include, but are not limitedto, modified or derivatized natural aminoacyl tRNA synthetases from oneor more of plants, animals, microorganisms, prokaryotes, eukaryotes,protozoa, bacteria, mammals, yeast, E. coli, or humans. In someembodiments, unnatural aminoacyl tRNA synthetases may include, but arenot limited to, aminoacyl tRNA synthetases with altered aminoacylspecificity and/or altered tRNA specificity, and/or altered editingability

As used herein, the term “altered specificity” means that thespecificity typically observed in nature has been changed. In someembodiments, altered specificity includes, but is not limited to,broadening the specificity to include, for example, recognition ofadditional amino acids, and/or additional tRNA. In some embodiments,altered specificity includes, but is not limited to, changing theidentity of the aminoacyl group and/or tRNA from the aminoacyl groupand/or tRNA recognized in nature.

Modified aminoacyl tRNA synthetases are known in the art, and includebut are not limited to, aminoacyl tRNA synthetases with relaxedsubstrate specificity through active site mutations as well as aminoacyltRNA synthetases with attenuated proofreading activity (e.g. Methods(2005) 36:227-238; Methods (2005) 36:291-298; Annu. Rev. Biochem. (2004)73:147-176; Science (2003) 301:964-967; Microbiology and MolecularBiology Reviews (2000) 64:202-236).

As used herein, the term “biological assembler or biological assemblers”means any mechanism that utilizes one or more biological components tosynthesize one or more peptides/proteins. In some embodiments,biological assemblers are peptide/protein assemblers. In someembodiments, biological assemblers are partially or completely isolated,purified, or separated from cells, other cellular material, and/ortissues. In some embodiments, biological assemblers are encompassed by asemi-permeable membrane and/or membrane-bound. In some embodiments,biological assemblers are modified, non-natural or recombinant. In someembodiments, biological assemblers include, but are not limited to, oneor more of ribosome-based assemblers and nonribosome-based assemblers.

As used herein, the term “ribosome-based assemblers” means biologicalassemblers that include, but are not limited to, one or more ribosomes.The ribosomes may be one or more of eukaryotic ribosomes and/orprokaryotic ribosomes. In some embodiments, the ribosome-basedassemblers are partially or completely isolated, purified, or separatedfrom cells, other cellular material, and/or tissues. In someembodiments, the ribosomes are from mitochondria and/or chloroplasts.The ribosomes may be from one or more of plants, animals,microorganisms, prokaryotes, eukaryotes, protozoa, bacteria, mammals,yeast, E. coli, and/or humans.

As used herein, the term “nonribosome-based assemblers” means biologicalassemblers that do not include ribosomes. In some embodiments, thenonribosome-based assemblers use one or more elements of a modularenzyme complex in which there is a common core structure, and optionallyinclude one or more different modules to perform additionalmanipulations on the evolving product. In some embodiments, thenonribosome-based assemblers are partially or completely isolated,purified, or separated from one or more of one or more unicellularorganisms, one or more plants, or one or more fungi.

As used herein, the term “biological assembler components or componentsof the biological assemblers” means one or more biological elements,and/or one or more non-biological elements, that make up the biologicalassemblers. In some embodiments, the components of one or morebiological assemblers include, but are not limited to, one or more ofone or more ribosomes, one or more ribosome subunits, one or moreribosomal RNA (rRNA) molecules, one or more protein molecules, one ormore translation factors, one or more enzymes, one or more energysources or one or more molecular chaperones. In some embodiments,biological assembler components include, but are not limited to, one ormore of one or more elements of a modular enzyme complex or one or moreadditional enzyme modules. In some embodiments, the components include,but are not limited to, one or more of one or more prokaryoticcomponents, one or more eukaryotic components, one or more mitochondrialcomponents, and/or one or more chloroplastic components.

Methods of partially and/or completely purifying or isolating natural,non-natural, and/or recombinant components of biological assemblers,including both ribosomal and non-ribosomal components, and re-assemblingfunctional peptide/protein synthetic machinery are known in the art(e.g. Methods (2005) 36:279-290; Methods (2005) 36:299-304; PNAS (2003)100:6353-6357; Nature (2001) 19:751-755; Royal Society of Chemistry(2004) 33:422-430). Methods for partially or completely encapsulatingisolated and/or purified biological assemblers and/or biologicalassembler components within natural or artificial semi-permeablemembranes, or partially or completely integrating isolated and/orpurified biological assemblers and/or biological assembler componentswithin natural or artificial semi-permeable membranes are known in theart (e.g. Cell (1997) 89:523-533; J. Cell Biology (1973) 56:191-205; J.Biol. Chem. (2000) 43:33820-33827).

As used herein, the term “components of peptide and/or target synthesis”or “peptide and/or target synthesis components” or the equivalent meansone or more biological components that may be optionally included in oneor more of the aspects described herein. As an example, peptidesynthesis components may include, but are not limited to, one or morebiological assemblers, one or more biological assembler components, oneor more charged tRNA, and/or one or more nucleic acids. Peptidesynthesis components may also include, but are not limited to, one ormore tRNA, one or more amino acids, one or more tRNA chargingcomponents, one or more nucleic acids, and one or more nucleic acidcharging components.

In one aspect, the disclosure is drawn to methods for peptide synthesis.Some methods comprise sequentially providing two or more charged tRNA toone or more identifiable locations. Some methods comprise co-localizingsequentially two or more charged tRNA with one or more biologicalassemblers. Some methods comprise co-localizing sequentially one or morebiological assemblers with two or more charged tRNA, wherein at leasttwo of the two or more tRNA are located at one or more differentlocations. Some methods comprise assembling a target peptide byco-localizing sequentially one or more biological assemblers and two ormore charged tRNA.

In some embodiments, one or more methods include synthesizing a targetpeptide by providing two or more charged tRNA to one or moreidentifiable locations. In some embodiments, one or more methods includesynthesizing a target peptide by co-localizing sequentially two or morecharged tRNA with one or more biological assemblers. In someembodiments, one or more methods include synthesizing a target peptideby co-localizing sequentially one or more biological assemblers with twoor more charged tRNA, wherein at least two of the two or more chargedtRNA are at one or more different locations.

Some embodiments include one or more methods of extra-cellular peptidesynthesis comprising co-localizing sequentially two or more charged tRNAwith one or more biological assemblers in vitro. Some embodimentsinclude one or more methods of extra-cellular peptide synthesiscomprising sequentially providing two or more charged tRNA to one ormore identifiable locations at one or more first identifiable timeintervals in vitro. Some embodiments include one or more methods ofextra-cellular peptide synthesis comprising co-localizing sequentiallyone or more biological assemblers with two or more charged tRNA, whereinat least two of the two or more charged tRNA are located at one or moredifferent locations in vitro. Some embodiments include one or moremethods of extra-cellular peptide synthesis comprising assembling atarget peptide in vitro by co-localizing sequentially one or morebiological assemblers, and two or more charged tRNA. In someembodiments, the one or more methods are cell-free.

In some embodiments, one or more methods include determining and/orselecting an assembly order; and co-localizing two or more charged tRNAwith one or more biological assemblers based on the assembly order. Insome embodiments, one or more methods include determining and/orselecting an assembly order; and providing two or more charged tRNA toone or more identifiable locations at one or more first identifiabletime intervals based on the assembly order. In some embodiments, one ormore methods include determining and/or selecting an assembly order; andco-localizing one or more biological assemblers with two or more chargedtRNA based on the assembly order, wherein at least two of the two ormore charged tRNA are located at one or more different locations. Insome embodiments, one or more methods include determining and/orselecting an assembly order; and assembling a target peptide byco-localizing one or more biological assemblers, and two or more chargedtRNA.

As used herein, the term “assembly order” means the process (orsequence) by which the one or more components of target peptidesynthesis are provided and/or co-localized and then optionally removedand/or separated.

In some embodiments, one or more methods further comprise eliminatingand/or removing and/or separating, optionally sequentially, one or morecomponents of peptide synthesis, optionally including, but not limitedto, one or more biological assemblers, one or more nucleic acids, one ormore biological assembler components, one or more charged tRNA, and/orone or more tRNA. In some embodiments, one or more methods furthercomprise consuming, optionally sequentially, two or more charged tRNA.In some embodiments, one or more methods further comprise eliminatingand/or removing and/or separating, optionally sequentially, two or morecharged tRNA and/or one or more released tRNA. In some embodiments, oneor more methods further include separating, optionally sequentially, oneor more biological assemblers from two or more charged tRNA and/or oneor more released tRNA, wherein at least two of the two or more chargedtRNA and/or released tRNA are located at one or more differentlocations. In some embodiments, one or more methods further includeseparating, optionally sequentially, one or more biological assemblersand two or more charged tRNA and/or one or more released tRNA.

As used herein, the term “co-localizing or providing or assembling”means any process resulting in one or more components being in the sameplace at the same time. By “in the same place at the same time” is meantphysical proximity such that the one or more components are capable ofinteraction on a molecular level. Co-localizing may include,commingling, combining, mixing, assembling, aggregating, injecting, orother similar processes.

As used herein, the term “synthesizing” means any process resulting inone or more components being combined and/or added to a prior component.For example, a process that results in combining two or more amino acidsto form a peptide, or a process that results in combining two or morenucleotides to form a nucleic acid.

As used herein, the term “removing or eliminating or separating” meansone or more processes that result in one or more peptide synthesiscomponents being no longer located in the same place. In someembodiments, one or more peptide synthesis components are at leastpartially removed and/or eliminated and/or consumed and/or separated. Insome embodiments, one or more components are moved to another location.

In illustrative embodiments, charged tRNA are at least partiallyconsumed when the attached amino acid is donated to the nascentpolypeptide. In some embodiments, charged tRNA not incorporated into thenascent polypeptide may be partially or completely removed (and/orseparated and/or eliminated) from one or more locations. In someembodiments, tRNA remaining after the previously attached amino acid isdonated to the nascent polypeptide are partially or completely removedand/or eliminated from one or more locations. In illustrativeembodiments, one or more charged tRNA and/or one or more tRNA areseparated from one or more biological assemblers. In illustrativeembodiments, one or more biological assemblers are separated from one ormore charged tRNA and/or one or more tRNA. In illustrative embodiments,one or more biological assemblers are removed from one or morelocations.

As used herein, the term “sequentially” when modifying processes, suchas, the processes including, for example, co-localizing, providing,removing, and/or eliminating, means any process that includes a temporalaspect such that the process acts upon one or more components atsubsequent times. Sequentially may include, but is not limited to, anyprocess that acts upon one or more components in a defined order.Sequentially may include, but is not limited to, any process that actson one or more components one after another.

Generic processes useful for co-localizing, providing, eliminating,removing, separating and/or assembling, and including sequentialprocesses, are known in the art and include, but are not limited to, oneor more of automated methods, mechanical methods, computer and/orsoftware-controlled methods, and fluid flow. Fluid flow includes, but isnot limited to, nanofluidics and microfluidics. Nanofluidics andmicrofluidics include, but are not limited to, continuous flowmicrofluidics and digital microfluidics, and have been developed for usein biological systems (Annu. Rev. Fluid Mech. (2004) 36:381-411; Annu.Rev. Biomed. Eng. (2002) 4:261-86; Science (1988) 242:1162-1164, Rev.Mod. Phys. (2005) 77:977-1026).

In illustrative embodiments, fluid flow is used to “flow” charged tRNAinto association (co-localization) with biological assemblers and to“flow” excess charged tRNA and/or released tRNA out of association(co-localization) with biological assemblers. In illustrativeembodiments, fluid flow is used to sequentially “flow” one type ofcharged tRNA after another into association with biological assemblersand to sequentially “flow” one type of excess charged tRNA and/orreleased tRNA after another out of association with biologicalassemblers. In illustrative embodiments, fluid flow is used to “flow”biological assemblers into association (co-localization) with chargedtRNA and to “flow” biological assemblers away from excess charged tRNAand/or released tRNA. In illustrative embodiments, fluid flow is used tosequentially “flow” one or more biological assemblers to locationscontaining one type of charged tRNA after another and to sequentially“flow” biological assemblers away from one type of excess charged tRNAand/or released tRNA after another.

In illustrative embodiments, one or more charged tRNA are provided inthe order of a target peptide sequence with each subsequent charged tRNAbeing provided after the aminoacyl residue from the prior charged tRNAis incorporated into a nascent polypeptide. In illustrative embodiments,excess charged tRNA and/or released tRNA areremoved/eliminated/separated before subsequent charged tRNA areprovided.

In illustrative embodiments, one or more biological assemblers areco-localized with one or more charged tRNA in the order of a targetpeptide sequence with each subsequent co-localization occurring afterthe aminoacyl residue from the prior charged tRNA is incorporated into anascent polypeptide. In illustrative embodiments, biological assemblersare removed/separated from excess charged tRNA and/or released tRNAprior to co-localization with subsequent charged tRNA.

In some embodiments, one or more methods includes co-localizing and/orproviding and/or assembling, optionally sequentially, one or morepeptide synthesis components at one or more identifiable time intervals.In some embodiments, one or more methods include providing, optionallysequentially, two or more charged tRNA to one or more identifiablelocations at one or more first identifiable time intervals. In someembodiments, one or more methods include co-localizing, optionallysequentially, two or more charged tRNA with one or more biologicalassemblers at one or more first identifiable time intervals. In someembodiments, one or more methods include co-localizing sequentially oneor more biological assemblers with two or more charged tRNA at one ormore first identifiable time intervals, wherein at least two of the twoor more charged tRNA are located at one or more different locations. Insome embodiments, one or more methods include assembling a targetpeptide by co-localizing sequentially one or more biological assemblersand two or more charged tRNA at one or more first identifiable timeintervals.

In some embodiments, one or more methods further include separatingand/or removing and/or eliminating and/or consuming, optionallysequentially, one or more peptide synthesis components at one or moreidentifiable time intervals. In some embodiments, one or more methodsfurther include removing and/or separating, optionally sequentially, twoor more charged tRNA and/or one or more released tRNA from one or morebiological assemblers at one or more second identifiable time intervals.In some embodiments, one or more methods further include separating,optionally sequentially, one or more biological assemblers from two ormore charged tRNA and/or one or more released tRNA at one or more secondidentifiable time intervals, wherein at least two of the two or morecharged tRNA and/or released tRNA are located at one or more differentlocations. In some embodiments, one or more methods further includeassembling a target peptide by separating, optionally sequentially, oneor more biological assemblers and two or more charged tRNA and/or thereleased tRNA at one or more second identifiable time intervals.

As used herein, the term “identifiable time interval” means a discreteamount of time that is optionally knowable, determinable, and/orcalculable. The term “one or more identifiable time intervals”, is usedherein to indicate time intervals for one or more processes. The one ormore identifiable time intervals may be the same or different fordifferent processes and/or elements of processes. The one or moreidentifiable time intervals may be the same or different for synthesisof different target peptides. One of skill in the art is able todetermine appropriate one or more identifiable time intervals based onthe teachings herein and in the art. The one or more identifiable timeintervals may be designated “first”, “second”, “third”, “fourth”,“fifth”, “sixth”, “seventh”, “eighth”, “ninth”, “tenth”, and so on forclarity to indicate that the time interval may, or may not, be the sameas other time intervals. Labeling one or more time intervals with thesame numeral may indicate the same or similar time intervals unlesscontext indicates otherwise.

In some embodiments, one or more identifiable time intervals are atleast partially based on a predicted rate of incorporation of two ormore amino acids into one or more peptides. In some embodiments, one ormore identifiable time intervals are at least partially based on apredicted rate of activity of one or more biological assemblers. In someembodiments, one or more identifiable time intervals are at leastpartially based on a predicted rate of translocation of one or morenucleic acids. In some embodiments, one or more identifiable timeintervals are at least partially based on a predicted rate of release oftRNA.

In some embodiments, one or more first identifiable time intervalsand/or one or more second identifiable time intervals are fromapproximately 0.001 seconds to approximately 0.1 seconds. In someembodiments, one or more first identifiable time intervals and/or one ormore second identifiable time intervals are approximately 0.01 seconds.

Many aspects of biological peptide synthesis, both in cells and incell-free systems, have been studied using a variety of natural andunnatural components (Cell (2002) 108:557-572, Ann. Rev. Biochem. (2004)73:657-704, Methods (2005) 36:279-290, Methods (2005) 36:299-304), andprovide a basis for predicting appropriate time intervals for additionand removal of target components for peptide synthesis. Rates ofincorporation of a variety of natural, unnatural, and arbitrary aminoacids into nascent peptides are known in the art for a variety ofbiological systems, including but not limited to, eukaryotic andprokaryotic systems. The corresponding rate of release of the tRNAfollowing incorporation of the amino acyl residue has also been studied.The rate of activity of a variety of eukaryotic and prokaryotic cellsand cell-free systems for peptide synthesis is known in the art(Molecular Biology of the Cell (2002) 343-344, J. Mol. Biol.(1984):549-576, J. Mol. Biol. (1989) 209:65-77, Methods (2005)36:279-290). The rate of translocation of nucleic acids in these systemshas also been studied. The rate of in vivo ribosomal incorporation ofamino acids into a protein is primarily limited by elongation (dominatedby acquisition rate of the cognate charged tRNA) and (for polyribosomaltranslation) by ribosomal initiation (Genetics (1998) 149:37-44; Journalof Theoretical Biology (2006) 239:417-434).

In some embodiments, one or more identifiable time intervals may includefrom approximately 0.001 seconds to approximately 0.1 seconds. In one ormore embodiments, one or more identifiable time intervals may include,but are not limited to, from 0.001 to 0.1, from 0.005 to 0.1, from 0.01to 0.1, from 0.05 to 0.1, from 0.001 to 0.05, from 0.001 to 0.01, andfrom 0.001 to 0.005 seconds. In some embodiments, one or moreidentifiable time intervals may include approximately 0.01 seconds. Insome embodiments, one or more identifiable time intervals may include,but are not limited to, approximately 0.001, 0.005, 0.01, 0.05, and 0.1seconds.

In some illustrative embodiments, charged tRNA may be “flowed” atpredetermined time intervals. One or more of the time intervals may beof the same length, of different lengths, of arbitrary lengths, ofrandom lengths, of variable lengths, of fixed lengths, and/or ofsequential lengths. In some embodiments, the time interval isdetermined, partially or completely, by the length of time needed and/oruseful to incorporate each additional amino acid residue into thenascent polypeptide, and/or by the length of time needed and/or usefulto remove excess charged tRNA and/or released tRNA from association(co-localization) with the ribosomal assemblers. In some embodiments,the time interval is determined, partially or completely, by internaland/or external feedback. In some embodiments, the internal and/orexternal feedback is partially or completely, related to the length oftime needed to incorporate each additional amino acid residue into thenascent polypeptide and/or by the length of time needed and/or useful toremove excess charged tRNA and/or released tRNA from association(co-localization) with the ribosomal assemblers.

In some embodiments, one or more methods may further include, monitoringamino acid incorporation into one or more peptides, biological assembleractivity, nucleic acid translocation, and/or tRNA release. In someembodiments, one or more methods further include, monitoring presence orabsence, concentration, and/or composition of charged tRNA and/or tRNA.

Methods for measuring and/or monitoring amino acid incorporation intoone or more peptides, biological assembler activity, nucleic acidtranslocation, or tRNA release are known in the art and include, but arenot limited to spectroscopy, fluorescence spectroscopy, surface plasmonresonance imaging, nuclear magnetic resonance imaging, and/orimmunoassays. Methods for measuring presence or absence, concentration,and/or compositions of charged tRNA or tRNA are known in the art, andinclude, but are not limited to, spectroscopy, fluorescencespectroscopy, surface plasmon resonance imaging, nuclear magneticresonance imaging, and/or immunoassays.

In some embodiments, one or more methods may include co-localizingand/or providing and/or assembling, optionally sequentially, one or morepeptide synthesis components at one or more identifiable time intervals,wherein the one or more identifiable time intervals are at leastpartially based on measurements of amino acid incorporation into one ormore peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. In some embodiments, the one or moreidentifiable time intervals are at least partially based on measurementsof availability of one or more nucleic acid codons.

In some embodiments, one or more methods include providing, optionallysequentially, two or more charged tRNA to one or more identifiablelocations at one or more first identifiable time intervals, wherein theone or more first identifiable time intervals are at least partiallybased on, but are not limited to, amino acid incorporation into one ormore peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. In some embodiments, one or moremethods include co-localizing sequentially two or more charged tRNA withone or more biological assemblers at one or more first identifiable timeintervals, wherein the one or more first identifiable time intervals areat least partially based on, but are not limited to, amino acidincorporation into one or more peptides, biological assembler activity,nucleic acid translocation, and/or tRNA release. In some embodiments,one or more methods include co-localizing sequentially one or morebiological assemblers with two or more charged tRNA at one or more firstidentifiable time intervals, wherein at least two of the two or morecharged tRNA are located at one or more different locations, and whereinthe one or more first identifiable time intervals are at least partiallybased on, but are not limited to, amino acid incorporation into one ormore peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. In some embodiments, one or moremethods include assembling a target peptide by co-localizingsequentially one or more biological assemblers with two or more chargedtRNA at one or more first identifiable time intervals, and wherein theone or more first identifiable time intervals are at least partiallybased on, but are not limited to, amino acid incorporation into one ormore peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. In some embodiments, the one or morefirst identifiable time intervals are at least partially based onmeasurements of availability of one or more nucleic acid codons.

In some embodiments, one or more methods may include removing and/orseparating and/or consuming and/or eliminating, optionally sequentially,one or more peptide synthesis components at one or more identifiabletime intervals, wherein the one or more identifiable time intervals areat least partially based on measurements and/or monitoring of amino acidincorporation into one or more peptides, biological assembler activity,nucleic acid translocation, and/or tRNA release. In some embodiments,the one or more identifiable time intervals are at least partially basedon measurements and/or monitoring of availability of one or more nucleicacid codons.

In some embodiments, one or more methods further include removing and/orseparating, optionally sequentially, two or more charged tRNA and/or oneor more tRNA from one or more biological assemblers at one or moresecond identifiable time intervals, wherein the one or more secondidentifiable time intervals are at least partially based on, but are notlimited to, amino acid incorporation into one or more peptides,biological assembler activity, nucleic acid translocation, and/or tRNArelease. In some embodiments, one or more methods further includeremoving and/or separating one or more biological assemblers from two ormore charged tRNA and/or one or more tRNA at one or more secondidentifiable time intervals, wherein the two or more charged tRNA arelocated at one or more different locations, and wherein the one or moresecond identifiable time intervals are at least partially based on, butare not limited to, amino acid incorporation into one or more peptides,biological assembler activity, nucleic acid translocation, and/or tRNArelease. In some embodiments, one or more methods further includeremoving and/or separating one or more biological assemblers and two ormore charged tRNA and/or one or more tRNA at one or more secondidentifiable time intervals, and wherein the one or more secondidentifiable time intervals are at least partially based on, but are notlimited to, amino acid incorporation into one or more peptides,biological assembler activity, nucleic acid translocation, and/or tRNArelease. In some embodiments, the one or more second identifiable timeintervals are at least partially based on measurements of availabilityof one or more nucleic acid codons.

In some embodiments, one or more methods may include co-localizingand/or providing and/or assembling, optionally sequentially, one or morepeptide synthesis components at one or more identifiable time intervals,wherein the one or more identifiable time intervals are at leastpartially based on measurements of concentrations of charged tRNA and/orreleased tRNA. In some embodiments, the one or more identifiable timeintervals are at least partially based on measurements of presence orabsence of one or more charged tRNA and/or one or more tRNA. In someembodiments, the one or more identifiable time intervals are at leastpartially based on measurements of presence or absence of one or moreanti-codons on one or more charged tRNA and/or one or more tRNA.

In some embodiments, one or more methods include providing, optionallysequentially, two or more charged tRNA at one or more identifiablelocations at one or more first identifiable time intervals, wherein theone or more first identifiable time intervals are at least partiallybased on measurements of concentrations of, presence or absence of,and/or composition of one or more charged tRNA and/or one or morereleased tRNA. In some embodiments, one or more methods includeco-localizing sequentially two or more charged tRNA with one or morebiological assemblers at one or more first identifiable time intervals,wherein the one or more first identifiable time intervals are at leastpartially based on measurements of concentrations of, presence orabsence of, and/or composition of one or more charged tRNA and/or one ormore released tRNA. In some embodiments, one or more methods includeco-localizing sequentially one or more biological assemblers with two ormore charged tRNA at one or more first identifiable time intervals,wherein at least two of the two or more charged tRNA are located at oneor more different locations, and wherein the one or more firstidentifiable time intervals are at least partially based on measurementsof concentrations of, presence or absence of, and/or composition of oneor more charged tRNA and/or one or more released tRNA. In someembodiments, one or more methods include assembling a target peptide byco-localizing sequentially one or more biological assemblers with two ormore charged tRNA at one or more first identifiable time intervals, andwherein the one or more first identifiable time intervals are at leastpartially based on measurements of concentrations of, presence orabsence of, and/or composition of one or more charged tRNA and/or one ormore released tRNA. In some embodiments, the one or more firstidentifiable time intervals are at least partially based on the presenceor absence of one or more anti-codons on one or more charged tRNA and/orreleased tRNA.

In some embodiments, one or more methods may further include removingand/or separating and/or consuming and/or eliminating, optionallysequentially, one or more peptide synthesis components at one or moreidentifiable time intervals, wherein the one or more identifiable timeintervals are at least partially based on measurements of concentrationsof charged tRNA or released tRNA. In some embodiments, the one or moreidentifiable time intervals are at least partially based on measurementsof presence or absence of one or more of the two or more charged tRNA orof the one or more tRNA. In some embodiments, the one or moreidentifiable time intervals are at least partially based on measurementsof presence or absence of one or more anti-codons on one or more of thetwo or more charged tRNA or the one or more tRNA.

In some embodiments, one or more methods further include removing and/orseparating, optionally sequentially, one or more charged tRNA and/or oneor more tRNA from one or more biological assemblers at one or moresecond identifiable time intervals, wherein the one or more secondidentifiable time intervals are at least partially based on measurementsof concentrations of, presence or absence of, and/or composition of oneor more charged tRNA and/or released tRNA. In some embodiments, one ormore methods further include removing and/or separating, optionallysequentially, one or more biological assemblers from two or more chargedtRNA at one or more second identifiable time intervals, wherein at leasttwo of the two or more charged tRNA are located at one or more differentlocations, and wherein the one or more second identifiable timeintervals are at least partially based on measurements of concentrationsof, presence or absence of, and/or composition of one or more chargedtRNA and/or released tRNA. In some embodiments, one or more methodsfurther include separating, optionally sequentially, one or morebiological assemblers and two or more charged tRNA at one or more secondidentifiable time intervals, and wherein the one or more secondidentifiable time intervals are at least partially based on measurementsof concentrations of, presence or absence of, and/or composition of oneor more charged tRNA and/or released tRNA. In some embodiments, the oneor more second identifiable time intervals are at least partially basedon the presence or absence of one or more anti-codons on one or morecharged tRNA and/or released tRNA.

In some embodiments, one or more methods may include one or moreidentifiable time intervals. Such identifiable time intervals mayinclude for example, but are not limited to, one or more of one or morefirst identifiable time intervals, one or more second identifiable timeintervals, one or more third identifiable time intervals, one or morefourth identifiable time intervals, one or more fifth identifiable timeintervals, and/or one or more sixth identifiable time intervals. In someembodiments, one or more identifiable time intervals may be the same asone or more other identifiable time intervals. In some embodiments, oneor more of identifiable time intervals may be different from one or moreother identifiable time intervals. In some embodiments, eachidentifiable time interval is determined separately.

In illustrative embodiments, one or more methods may include a firstidentifiable time interval for the (optionally sequential)co-localization of biological assemblers and charged tRNA. The one ormore methods may also include a second identifiable time interval forthe removal and/or separation (optionally sequential) of the biologicalassemblers from charged tRNA. These time intervals may be the same ordifferent.

In illustrative embodiments, one or more methods may include a firstidentifiable time interval for the (optionally sequential)co-localization of charged tRNA with biological assemblers. The one ormore methods may also include a second identifiable time interval forthe removal and/or separation (optionally sequential) of the chargedtRNA from the biological assemblers. These time intervals may be thesame or different. The one or more methods may also include athird/fourth/fifth identifiable time interval for the co-localization ofthe biological assemblers and/or biological assembler components and/ornucleic acids, for example, at an identifiable location. These timeintervals may all be the same as each other, or one or more may bedifferent. In some embodiments, one or more of these time intervals willbe different from the first and/or second identifiable time intervals.

In some embodiments, one or more methods include providing and/orco-localizing two or more charged tRNA, wherein two or more of the twoor more charged tRNA have the same anti-codon and are optionally chargedwith different amino acids. In some embodiments, one or more methodsinclude providing and/or co-localizing two or more charged tRNA, whereintwo or more of the two or more charged tRNA are optionally charged withthe same amino acids and have different anti-codons. In someembodiments, one or more methods include co-localizing one or morebiological assemblers with two or more charged tRNA, wherein at leasttwo of the two or more charged tRNA are at one or more differentlocations, and wherein two or more of the two or more charged tRNA havethe same anti-codon and are optionally charged with different aminoacids. In some embodiments, one or more methods include co-localizingone or more biological assemblers with two or more charged tRNA, whereinat least two of the two or more charged tRNA are at one or moredifferent locations, and wherein two or more of the two or more chargedtRNA are optionally charged with the same amino acids and have differentanti-codons. In some embodiments, one or more methods includeco-localizing one or more biological assemblers and two or more chargedtRNA, wherein two or more of the two or more charged tRNA have the sameanti-codon and are optionally charged with different amino acids. Insome embodiments, one or more methods include co-localizing one or morebiological assemblers and two or more charged tRNA, wherein two or moreof the two or more charged tRNA are optionally charged with the sameamino acids and have different anti-codons.

In illustrative embodiments, two of the charged tRNA may havetraditional stop anti-codons, for example AUU, but one may be chargedwith glycine and the other with methionine, for example. In illustrativeembodiments, two of the charged tRNA may have traditional stopanti-codons, for example one may have AUU and the other AUC, but bothtRNA may be charged with glycine, for example. In illustrativeembodiments, two of the charged tRNA may have traditional cysteineanti-codons, for example ACA, but one may be charged with glycine andthe other with methionine, for example. In illustrative embodiments, oneof the two charged tRNA may have a traditional cysteine anti-codon, forexample ACA, while the other may have a traditional histidineanti-codon, for example GUA, but both tRNA may be charged with glycine,for example.

In some embodiments, one or more methods comprises sequentiallyco-localizing and/or providing two or more charged anti-stop codon tRNAto one or more identifiable locations. In some embodiments, one or moremethods comprises sequentially providing two or more charged anti-stopcodon tRNA to one or more biological assemblers. In some embodiments,one or more methods comprises sequentially co-localizing one or morebiological assemblers with two or more charged anti-stop codon tRNA,wherein at least two charged tRNA are located at one or more differentlocations. In some embodiments, one or more methods comprisessequentially co-localizing one or more biological assemblers and two ormore charged anti-stop codon tRNA.

In some embodiments, one or more methods include 3 or more, 4 or more, 5or more, 6 or more, 8 or more, 10 or more, 15 or more, 20 or more, or 50or more charged anti-stop codon tRNA. In some embodiments, one or moremethods include from 2 to 500, 2 to 200, 2 to 100, 2 to 50, 2 to 25, 2to 10, 2 to 5, 4 to 500, 4 to 250, 4 to 100, 4 to 50, 4 to 25, 4 to 10,10 to 500, 10 to 250, 10 to 100, 10 to 50, 10 to 25, 20 to 500, 20 to250, 20 to 100, or 20 to 50 charged anti-stop codon tRNA.

In some embodiments, the two or more charged anti-stop codon tRNA havetwo different anti-stop codon recognition sites. In some embodiments,the two or more charged anti-stop codon tRNA with two differentanti-stop codon recognition sites are sequentially co-localized and/orprovided in an alternating anti-stop codon recognition site sequence. Insome embodiments, biological assemblers are sequentially co-localizedwith the two or more charged anti-stop codon tRNA in an alternatinganti-stop codon recognition site sequence. In some embodiments, thecharged anti-stop codon tRNA with two different anti-stop codonrecognition sites optionally have aminoacyl groups that changeirrespective and/or unrelated to of the identity of the tRNA anti-stopcodon. In some embodiments, the method includes at least 2, at least 3,at least 4, at least 5, at least 6, at least 8, at least 10, at least15, at least 20, or at least 25 charged anti-stop codon tRNA having twodifferent anti-stop codon recognition sites. In some embodiments, themethod includes from 2 to 500, 2 to 200, 2 to 100, 2 to 50, 2 to 25, 2to 10, 2 to 5, 4 to 500, 4 to 250, 4 to 100, 4 to 50, 4 to 25, 4 to 10,10 to 500, 10 to 250, 10 to 100, 10 to 50, 10 to 25, 20 to 500, 20 to250, 20 to 100, or 20 to 50 charged anti-stop codon tRNA having twodifferent anti-stop codon recognition sites.

In some embodiments, one or more methods include sequentiallyco-localizing and/or providing three or more charged anti-stop codontRNA having three different anti-stop codon recognition sites in arepeating anti-stop codon recognition site sequence. In someembodiments, one or more methods include sequentially co-localizing oneor more biological assemblers with three or more charged anti-stop codontRNA having three different anti-stop codon recognition sites in arepeating anti-stop codon recognition site sequence, wherein at leasttwo charged tRNA are located at one or more different locations. In someembodiments, one or more methods include sequentially co-localizing oneor more biological assemblers and three or more charged anti-stop codontRNA having three different anti-stop codon recognition sites in arepeating anti-stop codon recognition site sequence.

In some embodiments, the three or more charged anti-stop codon tRNA withthree different anti-stop codon recognition sites, optionally haveaminoacyl groups that change irrespective and/or unrelated to of theidentity of the tRNA anti-stop codon. In some embodiments, one or moremethods include at least 3, at least 4, at least 5, at least 6, at least8, at least 10, at least 15, at least 20, or at least 25 chargedanti-stop codon tRNA having three different anti-stop codon recognitionsites in a repeating anti-stop codon recognition site sequence. In someembodiments, one or more methods include 2 to 500, 2 to 200, 2 to 100, 2to 50, 2 to 25, 2 to 10, 2 to 5, 4 to 500, 4 to 250, 4 to 100, 4 to 50,4 to 25, 4 to 10, 10 to 500, 10 to 250, 10 to 100, 10 to 50, 10 to 25,20 to 500, 20 to 250, 20 to 100, or 20 to 50 charged anti-stop codontRNA having three different anti-stop codon recognition sites in arepeating anti-stop codon recognition site sequence.

In some embodiments, one or more methods include sequentially providingtwo or more charged tRNA in a target sequence to one or moreidentifiable locations. In some embodiments, one or more methods includesequentially co-localizing two or more charged tRNA with one or morebiological assemblers in a target sequence. In some embodiments, one ormore methods include co-localizing sequentially one or more biologicalassemblers with two or more charged tRNA in a target sequence, whereinat least two of the two or more charged tRNA are at one or moredifferent locations. In some embodiments, one or more methods includeco-localizing sequentially one or more biological assemblers and two ormore charged tRNA in a target sequence.

In some embodiments, one or more methods further include determining thetarget sequence for sequentially providing two or more charged tRNA toone or more identifiable locations. In some embodiments, one or moremethods further include determining the target sequence for sequentiallyco-localizing two or more charged tRNA with one or more biologicalassemblers. In some embodiments, one or more methods further includedetermining the target sequence for co-localizing sequentially the oneor more biological assemblers with the two or more charged tRNA, whereinat least two of the two or more charged tRNA are at one or moredifferent locations. In some embodiments, one or more methods furtherinclude determining the target sequence for co-localizing sequentiallythe one or more biological assemblers and the two or more charged tRNA.

In some embodiments, the target sequence is determined based on criteriaincluding, but not limited to, a target peptide sequence, a nucleic acidprotein coding sequence, a biological assembler, and/or one or morebiological assembler components. In some embodiments, the targetsequence is determined based on criteria including, but not limited to,user designations, target output, computer predictions, availability,predicted synthetic time, and/or cost.

In some embodiments, one or more methods include providing and/orco-localizing two or more charged tRNA sequentially at one or moreidentifiable locations at one or more identifiable time intervals,wherein the one or more identifiable locations contain one or morebiological assemblers and/or one or more nucleic acids. In someembodiments, the one or more nucleic acids have a defined and/or targetand/or selected protein coding sequence.

In some embodiments, one or more methods include co-localizingsequentially two or more charged tRNA with one or more peptideassemblers, one or more ribosome-based assemblers, one or morenonribosome-based assemblers, one or more prokaryotic ribosome-basedassemblers, one or more eukaryotic ribosome-based assemblers, one ormore E. coli ribosome-based assemblers, and/or one or more mitochondrialribosome-based assemblers. In some embodiments, one or more methodsinclude co-localizing sequentially one or more biological assemblers,one or more ribosome-based assemblers, one or more nonribosome-basedassemblers, one or more prokaryotic ribosome-based assemblers, one ormore eukaryotic ribosome-based assemblers, one or more E. coliribosome-based assemblers, and/or one or more mitochondrialribosome-based assemblers with two or more charged tRNA, wherein atleast two of the two or more charged tRNA are at one or more differentlocations. In some embodiments, one or more methods includeco-localizing sequentially one or more biological assemblers, one ormore ribosome-based assemblers, one or more nonribosome-basedassemblers, one or more prokaryotic ribosome-based assemblers, one ormore eukaryotic ribosome-based assemblers, one or more E. coliribosome-based assemblers, and/or one or more mitochondrialribosome-based assemblers and two or more charged tRNA.

In some embodiments, one or more methods include providing one or morefirst charged tRNA to one or more identifiable locations; providing oneor more second charged tRNA to the one or more identifiable locations;and optionally repeating. In some embodiments, one or more methodsfurther comprise providing one or more third charged tRNA to one or moreidentifiable locations. In some embodiments, one or more methodscomprises co-localizing one or more first charged tRNA with one or morebiological assemblers; co-localizing one or more second charged tRNAwith one or more biological assemblers; and optionally repeating. Insome embodiments, one or more methods further comprise co-localizing oneor more third charged tRNA with one or more biological assemblers. Insome embodiments, one or more methods include co-localizing one or morebiological assemblers with one or more first charged tRNA at one or morefirst locations; co-localizing the one or more biological assemblerswith one or more second charged tRNA at one or more second locations;and optionally repeating. In some embodiments, one or more methodsfurther include co-localizing one or more biological assemblers with oneor more third charged tRNA at one or more third locations. In someembodiments, one or more methods include co-localizing one or morebiological assemblers and one or more first charged tRNA at one or morefirst locations; co-localizing the one or more biological assemblers andone or more second charged tRNA at one or more second locations; andoptionally repeating. In some embodiments, one or more methods furtherinclude co-localizing one or more biological assemblers and one or morethird charged tRNA at one or more third locations.

In some embodiments, one or more methods further comprise providing oneor more additional charged tRNA to one or more identifiable locations.In some embodiments, one or more methods further comprise co-localizingone or more additional charged tRNA with one or more biologicalassemblers. In some embodiments, one or more methods further compriseco-localizing one or more biological assemblers with one or moreadditional charged tRNA, wherein the one or more additional charged tRNAare optionally at one or more different locations. In some embodiments,one or more methods further comprise co-localizing one or morebiological assemblers and one or more additional charged tRNA.

In some embodiments, the one or more first charged tRNA is optionallythe same as and/or optionally different from the one or more secondcharged tRNA. In some embodiments, the one or more first charged tRNAincludes a stop codon recognition site, and the one or more secondcharged tRNA includes a stop codon recognition site. In someembodiments, the one or more first charged tRNA stop codon recognitionsite is different from the one or more second charged tRNA stop codonrecognition site. In some embodiments, the one or more third chargedtRNA includes a stop codon recognition site that is optionally the sameas the one or more first charged tRNA stop codon recognition site and/orthe one or more second charged tRNA stop codon recognition site, oroptionally different from the one or more first charged tRNA stop codonrecognition site and/or the one or more second charged tRNA stop codonrecognition site. In some embodiments, the one or more additionalcharged tRNA have stop codon recognition sites. In some embodiments, theone or more additional charged tRNA having stop codon recognition sitesare co-localized such that the stop codon recognition sites of thecharged tRNA alternate. In some embodiments, the one or more biologicalassemblers are co-localized with the one or more additional charged tRNAhaving stop codon recognition sites such that the stop codon recognitionsites of the charged tRNA alternate.

In some embodiments, the one or more first charged tRNA, the one or moresecond charged tRNA, the one or more third charged tRNA, and/or the oneor more additional charged tRNA are optionally the same as, oroptionally different from, each other. In some embodiments, the tRNAportion of one or more of the one or more first charged tRNA, the one ormore second charged tRNA, the one or more third charged tRNA, or the oneor more additional charged tRNA are optionally the same as, oroptionally different from, each other. In some embodiments, one or moreof the anticodon portions of one or more of the one or more firstcharged tRNA, the one or more second charged tRNA, the one or more thirdcharged tRNA, or the one or more additional charged tRNA are one or morestop codon recognition sites. In some embodiments, the amino acidportion of one or more of the one or more first charged tRNA, the one ormore second charged tRNA, the one or more third charged tRNA, or the oneor more additional charged tRNA are optionally the same as, oroptionally different from, each other.

In some embodiments, one or more methods comprises co-localizing one ormore first charged tRNA with one or more biological assemblers, the oneor more first charged tRNA charged with one or more first arbitraryamino acid; removing one or more first tRNA, the one or more first tRNAreleased during peptide synthesis; co-localizing one or more secondcharged tRNA with one or more biological assemblers, the one or moresecond charged tRNA charged with one or more second arbitrary aminoacid; removing one or more second tRNA, the one or more second tRNAreleased during peptide synthesis; and optionally repeating. In someembodiments, the method includes co-localizing one or more biologicalassemblers with one or more first charged tRNA in one or more firstlocations, the one or more first charged tRNA charged with one or morefirst arbitrary amino acid; removing one or more first tRNA, the one ormore first tRNA released during peptide synthesis; co-localizing one ormore biological assemblers with one or more second charged tRNA at oneor more second locations, the one or more second charged tRNA chargedwith one or more second arbitrary amino acid; removing one or moresecond tRNA, the one or more second tRNA released during peptidesynthesis; and optionally repeating.

In some embodiments, one or more methods include co-localizing one ormore biological assemblers with one or more first charged tRNA at one ormore first locations, the one or more first charged tRNA charged withone or more arbitrary amino acids; removing the one or more biologicalassemblers from the one or more first locations; co-localizing the oneor more biological assemblers with one or more second charged tRNA atone or more second locations, the second one or more charged tRNAcharged with one or more arbitrary amino acids; removing the one or morebiological assemblers from the one or more second locations; andoptionally repeating. In some embodiments, one or more methods includeco-localizing one or more biological assemblers and one or more firstcharged tRNA at one or more first locations, the first one or morecharged tRNA charged with one or more arbitrary amino acids; separatingthe one or more first charged tRNA and/or one or more released tRNA andthe one or more biological assemblers; co-localizing the one or morebiological assemblers and one or more second charged tRNA at one or moresecond locations, the second one or more charged tRNA charged with oneor more arbitrary amino acids; separating the one or more second chargedtRNA and/or one or more released tRNA and the one or more biologicalassemblers; and optionally repeating.

In some embodiments, the first arbitrary amino acid is optionally thesame as, or optionally different from, the second arbitrary amino acid.In some embodiments, the first tRNA is optionally the same as, oroptionally different from, the second tRNA. In some embodiments, thefirst charged tRNA is optionally the same as, or optionally differentfrom, the second charged tRNA. In some embodiments, the one or morefirst arbitrary amino acid is the same as the one or more secondarbitrary amino acid, and the one or more first tRNA is different fromthe one or more second tRNA. In some embodiments, the first and thesecond tRNA have different anti-codons. In some embodiments, the one ormore first tRNA is the same as the one or more second tRNA, and the oneor more first arbitrary amino acids are different from the one or moresecond arbitrary amino acids. In some embodiments, the first and thesecond tRNA have the same anti-codon.

In illustrative embodiments, one or more methods include: providing thefirst charged tRNA having an anti-codon that recognizes the first codonof the translatable reading frame and having an amino acid attached thatis the first amino acid of the target polypeptide; allowing sufficienttime for docking of the charged tRNA; providing the second charged tRNAhaving an anti-codon that recognizes the second codon of thetranslatable reading frame and having an amino acid attached that is thesecond amino acid of the target polypeptide; allowing sufficient timefor docking of the charged tRNA, peptide bond formation between thefirst two amino acids, and release of the first tRNA; removal of thefirst released tRNA; providing the third charged tRNA having ananti-codon that recognizes the third codon of the translatable readingframe and having an amino acid attached that is the third amino acid ofthe target polypeptide; allowing sufficient time for docking of thecharged tRNA, peptide bond formation between the second and the thirdamino acids, and release of the second tRNA; removal of the secondreleased tRNA; and repeating the process for the target peptidesequence.

In some embodiments, one or more of the methods described hereinincludes charging one or more tRNA with one or more arbitrary aminoacids, one or more natural amino acids or one or more unnatural aminoacids. In some embodiments, one or more of the methods described hereinincludes charging one or more anti-stop codon tRNA with one or morenatural amino acids or one or more unnatural amino acids. In someembodiments, aminoacylation is mediated by aminoacyl tRNA synthetasesincluding one or more natural and/or one or more unnatural aminoacyltRNA synthetases.

In some embodiments, one or more of the methods described hereinincludes selecting two or more charged tRNA. In some embodiments, two ormore charged tRNA are selected at least partially, or completely, basedon criteria including, but not limited to, target peptide sequence, anucleic acid protein coding sequence, a biological assembler, and/or oneor more biological assembler components. In some embodiments, theprotein coding region of the nucleic acid sequence includes one or morecodons selected from the group consisting of two or more stop codons, atleast three stop codons, two or more alternating stop codons, one ormore singlet codons, one or more doublet codons, one or more tripletcodons, one or more quadruplet codons, one or more quintuplet codons,and one or more sextuplet codons. In some embodiments, two or morecharged tRNA are selected based on criteria including, but not limitedto, user designations, target output, computer predictions,availability, predicted synthetic time, and/or cost.

In some embodiments, one or more of the methods described hereinincludes selecting one or more biological assemblers. In someembodiments, one or more biological assemblers are selected at leastpartially based on criteria including, but not limited to, one or moreof a target peptide sequence, one or more charged tRNA, or a nucleicacid protein coding sequence. In some embodiments, one or morebiological assemblers are selected at least partially based on criteriaincluding, but not limited to, user designations, target output,computer predictions, availability, predicted synthetic time, and cost.

In some embodiments, one or more of the methods described hereinincludes selecting one or more components of one or more biologicalassemblers. In some embodiments, one or more biological assemblercomponents are selected at least partially based on criteria including,but not limited to, one or more of a target peptide sequence, one ormore charged tRNA, or a nucleic acid protein coding sequence. In someembodiments, one or more biological assembler components are selected atleast partially based on criteria including, but not limited to, userdesignations, target output, computer predictions, availability,predicted synthetic time, and cost.

As used herein, the term “selecting” means any process used to identifyfor use one or more target components. Processes include, but are notlimited to, user selected, user identified, software method analysis,algorithm-based, computer mediated, operations research, optimization,simulation, queuing theory, and/or game theory.

In some embodiments, one or more methods include assembling and/orco-localizing and/or providing one or more components of one or morebiological assemblers. In some embodiments, one or more methods includeassembling one or more components of the one or more biologicalassemblers at one or more third identifiable time intervals. In someembodiments, the one or more third identifiable time intervals are atleast partially based on a predicted rate of incorporation of two ormore amino acids into one or more peptides, a predicted rate of activityof the one or more biological assemblers, a predicted rate oftranslocation of one of more nucleic acids, and/or a predicted rate ofrelease of tRNA. In illustrative embodiments, one or more methodsinclude assembling one or more first biological assembler components,assembling a target peptide, removing the one or more first biologicalassembler components, and co-localizing one or more second biologicalassembler components. In illustrative embodiments, one or more methodsinclude assembling one or more first biological assembler components,commencing synthesis of a target peptide, co-localizing one or moresecond biological assembler components, and assembling a target peptide.

In some embodiments, one or more methods include assembling one or morecomponents of one or more biological assemblers at one or more thirdidentifiable time intervals, and further comprises monitoring amino acidincorporation into one or more peptides, biological assembler activity,nucleic acid translocation, and/or tRNA release. In some embodiments,one or more methods include assembling one or more components of one ormore biological assemblers at one or more third identifiable timeintervals, and further include monitoring the presence or absence,concentration, and/or compositions of one or more charged tRNA and/orone or more tRNA. In some embodiments, the one or more identifiable timeintervals are at least partially based on availability of one or morenucleic acid codons. In some embodiments, the one or more identifiabletime intervals are at least partially based on the concentrations of oneor more charged tRNA and/or one or more tRNA. In some embodiments theone or more identifiable time intervals are based on the presence orabsence of one or more charged tRNA and/or one or more tRNA. In someembodiments, the one or more identifiable time intervals are based onthe presence or absence of one or more anti-codons on one or morecharged tRNA and/or one or more tRNA.

In some embodiments, one or more methods of co-localizing sequentiallytwo or more tRNA with one or more biological assemblers, further includeco-localizing the one or more biological assemblers at one or moreidentifiable locations. In some embodiments, one or more methods includeco-localizing one or more biological assemblers at one or moreidentifiable locations at one or more fourth identifiable timeintervals. In some embodiments, the one or more fourth identifiable timeintervals are at least partially based on a predicted rate ofincorporation of one or more amino acids into one or more peptides, apredicted rate of activity of one or more biological assemblers, apredicted rate of translocation of one or more nucleic acids, and/or apredicted rate of release of tRNA. In illustrative embodiments, one ormore methods include co-localizing one or more first biologicalassemblers, assembling a target peptide, removing the one or more firstbiological assemblers, and co-localizing one or more second biologicalassemblers.

In some embodiments, one or more methods includes co-localizing one ormore biological assemblers at one or more identifiable locations at oneor more fourth identifiable time intervals, and further comprisesmonitoring amino acid incorporation into one or more peptides,biological assembler activity, nucleic acid translocation, and/or tRNArelease. In some embodiments, the one or more identifiable timeintervals are based on the results of the monitoring. For example, insome embodiments the one or more identifiable time intervals are atleast partially based on measurements of amino acid incorporation intoone or more peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. In some embodiments, one oridentifiable time intervals are at least partially based on availabilityof one or more nucleic acid codons.

In some embodiments, one or more methods includes co-localizing one ormore biological assemblers at one or more identifiable locations at oneor more fourth identifiable time intervals, and further comprisesmonitoring the presence or absence, concentration, and/or compositionsof one or more charged tRNA and/or one or more tRNA released duringpeptide synthesis. In some embodiments, the one or more identifiabletime intervals are based on the results of monitoring. For example, theone or more identifiable time intervals may be at least partially basedon the concentrations of one or more charged tRNA and/or one or moretRNA, the presence or absence of one or more charged tRNA and/or one ormore tRNA, and/or the presence or absence of one or more anti-codons onone or more charged tRNA and/or one or more tRNA.

In some embodiments, co-localizing, optionally sequentially, two or morecharged tRNA with one or more biological assemblers occurs at leastpartially, and optionally completely, following co-localizing the one ormore biological assemblers at one or more identifiable locations. Insome embodiments, co-localizing, optionally sequentially, one or morebiological assemblers with two or more charged tRNA, wherein at leasttwo of the two or more charged tRNA are at different locations, occursat least partially, and optionally completely, following co-localizingthe two or more charged tRNA at one or more different locations.

As used herein, the term “assembling” means any process resulting in oneor more components being in the same place. In some embodiments, thecomponents may be assembled by one or more methods including, but notlimited to, one or more of co-localized, provided, injected, aggregated,commingled, combined, mixed or any other similar method.

In illustrative embodiments, fluid flow is used to “flow” one or morecomponents into association (or assemblage). In some embodiments, thecomponents may be all of one type, or of one or more types. In someembodiments, the components may be an admixture of ribosomal andnon-ribosomal components, or biological and non-biological components.

As used herein, the term “type” means a difference in kind. For example,“type” as used with biological assemblers and/or biological assemblercomponents may refer to chloroplast versus mitochondrial ribosomalassemblers, or prokaryotic versus eukaryotic ribosomal assemblers, orribosomal versus nonribosome assemblers. Type may also refer todifferences in kind for nucleic acids, for example, DNA versus RNA, oreukaryotic versus prokaryotic, or plasmid versus linear. Type may alsorefer to differences in kind for charged tRNA and/or tRNA, for example,based on differences in the anti-codon, or eukaryotic versusprokaryotic, or natural versus unnatural. Type may also refer todifferences in kind for amino acids, for example, based on differencesof natural versus unnatural.

In some embodiments, one or more methods include one or more biologicalassemblers that are affixed at one or more identifiable locations. Insome embodiments, one or more of the methods further comprise affixingone or more biological assemblers at one or more identifiable locationsand/or to one or more devices. In some embodiments, one or more of themethods include co-localizing sequentially two or more charged tRNA withone or more biological assemblers, wherein the one or more biologicalassemblers are affixed at one or more identifiable locations. In someembodiments, one or more methods include affixing one or more nucleicacids at one or more identifiable locations and/or to one or moredevices.

In some embodiments, one or more methods include two or more chargedtRNA affixed at one or more different locations. In some embodiments,one or more methods include affixing two or more charged tRNA at two ormore different locations and/or the two or more different devices. Insome embodiments, one or more methods include co-localizing sequentiallyone or more biological assemblers with two or more charged tRNA, whereinat least two of the two or more charged tRNA are located at one or moredifferent locations, and wherein the at least two of the two or morecharged tRNA are affixed at the two or more different locations. In someembodiments, one or more devices include, but are not limited to, one ormore microelectromechanical systems (MEMS) devices, beads, and/orimmunoassay arrays.

In illustrative embodiments, two or more charged tRNA are affixed in twoor more liquid beads. In illustrative embodiments, fluid flow is used toco-localize one or more biological assemblers sequentially with each ofthe two or more charged tRNA in two or more liquid beads. Inillustrative embodiments, excess charged tRNA and/or released tRNA areremoved using fluid flow and differential filtration.

In some embodiments, one or more methods include co-localizingsequentially one or more biological assemblers with two or more chargedtRNA, wherein at least two of the two or more charged tRNA are locatedat one or more different locations, and further includes co-localizingthe at least two of the two or more charged tRNA at the one or moredifferent locations. In some embodiments, the co-localizing sequentiallyone or more biological assemblers with the at least two of the two ormore charged tRNA occurs at least partially following the co-localizingthe at least two of the two or more charged tRNA at the one or moredifferent locations. In some embodiments, one or more methods includeco-localizing sequentially one or more biological assemblers with two ormore charged tRNA, wherein at least two of the two or more charged tRNAare located at one or more different locations, and further includesremoving one or more of the at least two of the two or more charged tRNAor one or more released tRNA from the one or more different locations.

In illustrative embodiments, one or more methods comprise:co-localizing, optionally sequentially, one or more first charged tRNAat one or more first identifiable locations, one or more second chargedtRNA at one or more second identifiable locations, and one or more thirdcharged tRNA at one or more third identifiable locations; co-localizingand subsequently removing one or more biological assemblers with the oneor more first charged tRNA at the one or more first locations, with theone or more second charged tRNA at the one or more second locations,and/or with the one or more third charged tRNA at the one or more thirdlocations in a target sequence; removing one or more first charged tRNAand/or one or more released tRNA from the one or more first locations,one or more second charged tRNA and/or one or more released tRNA fromthe one or more second locations, and/or one or more third charged tRNAand/or one or more released tRNA from at the one or more third locationsdepending on the target sequence; and optionally repeating until atarget peptide is synthesized.

In some embodiments, one or more methods include co-localizingsequentially one or more biological assemblers with two or more chargedtRNA, wherein at least two of the two or more charged tRNA are locatedat one or more different locations, and further includes co-localizingthe at least two of the two or more charged tRNA at the one or moredifferent locations at one or more identifiable time intervals. In someembodiments, one or more methods include co-localizing sequentially oneor more biological assemblers with two or more charged tRNA, wherein atleast two of the two or more charged tRNA are located at one or moredifferent locations, and further includes removing one or more of the atleast two of the two or more charged tRNA or one or more released tRNAfrom the one or more different locations at one or more identifiabletime intervals. The time intervals for providing the charged tRNA andthe time intervals for removing the charged tRNA and/or released tRNAmay be the same or different. The one or more identifiable timeintervals for providing the charged tRNA and the time intervals forremoving the charged tRNA and/or released tRNA are at least partiallybased on one or more of a predicted rate of incorporation of two or moreamino acids into one or more peptides, a predicted rate of activity ofthe one or more biological assemblers, a predicted rate of translocationof one or more nucleic acids, or a predicted rate of release of tRNA.

In some embodiments, one or more methods include co-localizingsequentially one or more biological assemblers with two or more chargedtRNA, wherein at least two of the two or more charged tRNA are locatedat one or more different locations; co-localizing the at least two ofthe two or more charged tRNA at the one or more different locations atone or more identifiable time intervals; and further includes monitoringamino acid incorporation into one or more peptides, biological assembleractivity, nucleic acid translocation, and/or tRNA release. In someembodiments, one or more methods include co-localizing sequentially oneor more biological assemblers with two or more charged tRNA, wherein atleast two of the two or more charged tRNA are located at one or moredifferent locations; removing one or more of the at least two of the twoor more charged tRNA or one or more released tRNA from the one or moredifferent locations at one or more identifiable time intervals; andfurther includes monitoring amino acid incorporation into one or morepeptides, biological assembler activity, nucleic acid translocation,and/or tRNA release. In some embodiments, the one or more firstidentifiable time intervals are at least partially based on the resultsof the monitoring, including but not limited to measurements of theamino acid incorporation into the one or more peptides, the biologicalassembler activity, the nucleic acid translocation, and/or the tRNArelease. In some embodiments, the one or more first identifiable timeintervals are at least partially based on availability of one or morenucleic acid codons.

In some embodiments, one or more methods include co-localizingsequentially one or more biological assemblers with two or more chargedtRNA, wherein at least two of the two or more charged tRNA are locatedat one or more different locations; co-localizing the at least two ofthe two or more charged tRNA at the one or more different locations atone or more identifiable time intervals; and further includes monitoringthe presence or absence, concentration, and/or composition of one ormore charged tRNA and/or one or more tRNA. In some embodiments, one ormore methods include co-localizing sequentially one or more biologicalassemblers with two or more charged tRNA, wherein at least two of thetwo or more charged tRNA are located at one or more different locations;removing one or more of the at least two of the two or more charged tRNAor one or more released tRNA from the one or more different locations atone or more identifiable time intervals; and further includes monitoringthe presence or absence, concentration, and/or composition of one ormore charged tRNA and/or one or more tRNA. In some embodiments, the oneor more first identifiable time intervals are at least partially basedon the results of the monitoring, including but not limited to theconcentration, presence or absence of one or more charged tRNA and/orone or more tRNA, and/or the presence or absence of one or moreanti-codons on one or more charged tRNA and/or one or more tRNA.

As used herein, the term “identifiable location” means a position inspace and time that can be determined. In some embodiments, one or moreidentifiable locations are internal to a device or apparatus and/orexternal to a device or apparatus. In some embodiments, the one or moreidentifiable locations are moving in time and/or moving in space. Insome embodiments, the movement in time and/or space may be one or moreof steady, fluctuating, predictable or other type of movement so long asthe location can be identified at a particular place and time.

In some embodiments, one or more of the processes and/or elements ofprocesses occur at one or more identifiable locations that may be thesame or may be different. In some embodiments the terms, “first”,“second”, “third”, “fourth”, “fifth”, “sixth”, etc. may be used toindicate that the identifiable locations are optionally differentidentifiable locations. Generally, identifiable locations indicated bythe same numeral are the same locations unless context indicatesotherwise.

As used herein, the term “different location” means an identifiablelocation that is in a different position in space and/or time fromanother identifiable location.

As used herein, the term “devices” means any configuration capable oflocalizing and/or containing one or more components at leasttemporarily. Devices may include, but are not limited to, containers,receptacles, semi-permeable membranes, beads of liquid, MEMS,microfluidics devices, arrays, liposomes, and/or surface-tensionattached liquids.

As used herein, the term “affixing” means any process that at leasttemporarily restricts the movement of one or more components in relationto an identifiable location and/or one or more devices. Processesinclude, but are not limited to, attachment, filtration,ultrafiltration, resins, changes in aperture diameter, optical traps,and/or electric fields. Affixing may be through direct and/or indirectmeans including, for example, affixing one or more biological assemblersby affixing the one or more nucleic acids that the one or morebiological assemblers are translating. Attachment includes, but is notlimited to, methods known in the art for attaching membranes to avariety of support structures, for attaching nucleic acids to a varietyof support structures, and for attaching proteins to a variety ofsupport structures. Support structures include, but are not limited to,beads, microfluidic devices, MEMS devices, carbon nanotubes, arrays,and/or microstructured surfaces.

In some embodiments, one or more methods include providing and/orco-localizing two or more charged tRNA sequentially into one or morereceptacles at one or more identifiable locations. In some embodiments,one or more methods include providing and/or co-localizing two or morecharged tRNA sequentially into one or more receptacles at one or moreidentifiable locations at one or more identifiable time intervals. Insome embodiments, one or more methods include injecting sequentially twoor more charged tRNA into one or more receptacles containing one or morebiological receptors. In some embodiments, the one or more receptaclescontain one or more biological assemblers and/or one or more nucleicacids.

In some embodiments, one or more methods include co-localizingsequentially two or more charged tRNA with one or more biologicalassemblers that are co-localized with one or more nucleic acids. In someembodiments, one or more methods include co-localizing sequentially oneor more biological assemblers and two or more charged tRNA, wherein theone or more biological assemblers are co-localized with one or morenucleic acids. In some embodiments, multiple biological assemblers areco-localized with one or more nucleic acids. In some embodiments,multiple biological assemblers are optionally translating one or morenucleic acids at the same time.

In some embodiments, the one or more nucleic acids include, but are notlimited to, one or more DNA, one or more cDNA, one or more RNA, and/orone or more mRNA. In some embodiments, one or more nucleic acids may berecombinant, circular, linear, plasmid, double stranded, singlestranded, poly-adenylated, or any other form known in the art suitablefor protein synthesis. In some embodiments, one more nucleic acids havea defined and/or selected and/or target protein coding sequence.

As used herein, the term “nucleic acid or nucleic acids” means one ormore complex, high-molecular-weight biochemical macromolecules composedof nucleotide chains. Nucleic acids include, but are not limited to, oneor more forms of deoxyribonucleic acid (DNA), ribonucleic acid (RNA;includes messenger RNA (mRNA)), and complementary DNA (cDNA; DNAsynthesized from an mRNA template).

As used herein, the term “target protein coding sequence” means atranslatable reading frame in one or more nucleic acids. In someembodiments, the translatable reading frames are open reading framesthat translate into a target peptide using the standard genetic code. Insome embodiments, the translatable reading frames do not translate intoa target peptide using the standard genetic code.

In some embodiments, the translatable reading frames contain at leastone stop codon. In some embodiments, the translatable reading frameshave a protein coding sequence having at least 2, at least 3, at least4, at least 5, at least 6, at least 8, at least 10, at least 15, atleast 20, or at least 25 stop codons. In some embodiments, thetranslatable reading frames have a protein coding sequence having from 2to 500, 2 to 200, 2 to 100, 2 to 50, 2 to 25, 2 to 10, 2 to 5, 4 to 500,4 to 250, 4 to 100, 4 to to 25, 4 to 10, 10 to 500, 10 to 250, 10 to100, 10 to 50, 10 to 25, 20 to 500, 20 to 250, 20 to 100, or 20 to 50stop codons.

In some embodiments, the stop codons are two different stop codons in analternating sequence. In some embodiments, the alternating stop codonsequence includes, but is not limited to, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 8, at least 10, at least 15,at least 20, or at least 25 alternating stop codons. In someembodiments, the alternating stop codon sequence has from 2 to 500, 2 to200, 2 to 100, 2 to 50, 2 to 25, 2 to 10, 2 to 5, 4 to 500, 4 to 250, 4to 100, 4 to 50, 4 to 25, 4 to 10, 10 to 500, 10 to 250, 10 to 100, 10to 50, 10 to 25, 20 to 500, 20 to 250, 20 to 100, or 20 to 50alternating stop codons.

In some embodiments, the stop codons are three different stop codons ina repeating sequence. In some embodiments, the repeating stop codonsequence includes, but is not limited to, at least 3, at least 4, atleast 5, at least 6, at least 8, at least 10, at least 15, at least 20,or at least 25 repeating stop codons. In some embodiments, the repeatingstop codon sequence has from 2 to 500, 2 to 200, 2 to 100, 2 to 50, 2 to25, 2 to 10, 2 to 5, 4 to 500, 4 to 250, 4 to 100, 4 to 50, 4 to 25, 4to 10, 10 to 500, 10 to 250, 10 to 100, 10 to 50, 10 to 25, 20 to 500,20 to 250, 20 to 100, or 20 to 50 repeating stop codons.

In some embodiments, the translatable reading frames contain one or moreof at least one singlet codon, at least one doublet codon, at least onetriplet codon, at least one quadruplet codon, at least one quintupletcodon, or at least one sextuplet codon. In some embodiments, thetranslatable reading frames have a protein coding sequence having atleast 2, at least 3, at least 4, at least 5, at least 6, at least 8, atleast 10, at least 15, at least 20, or at least 25 singlet, doublet,triplet, quadruplet, quintuplet, and/or sextuplet codons. In someembodiments, the translatable reading frames have a protein codingsequence having from 2 to 500, 2 to 200, 2 to 100, 2 to 50, 2 to 25, 2to 10, 2 to 5, 4 to 500, 4 to 250, 4 to 100, 4 to 50, 4 to 25, 4 to 10,10 to 500, 10 to 250, 10 to 100, 10 to 50, 10 to 25, 20 to 500, 20 to250, 20 to 100, or 20 to 50 singlet, doublet, triplet, quadruplet,quintuplet, and/or sextuplet codons.

Methods of synthesizing nucleic acids are known in the art including,but not limited to, chemical and enzymatic synthesis. Proteins/peptidestranslated from nucleic acids with modified translatable reading frames,including sense codon reassignment are known in the art (e.g. Methods(2005) 36:227-238; Methods (2005) 36:270-278; Methods (2005) 36:279-290;Methods (2005) 36:291-298; Annu. Rev. Biochem. (2004) 73:147-176;Nucleic Acids Research (2004) 32:6200-6211; PNAS (2003) 100:6353-6357)

As used herein, the term “target protein or target protein sequence”means one or more identified and/or selected polypeptide sequences. Insome embodiments, the target peptide sequence is directly translatablefrom the target protein coding sequence of one or more nucleic acidsusing the genetic code. In some embodiments, the target peptide sequenceis not directly translatable from the target protein coding sequence ofone or more nucleic acids using the genetic code.

In other embodiments, one or more methods include co-localizing one ormore nucleic acids with one or more biological assemblers. Someembodiments include any process that results in one or more componentsbeing in the same place. One or more nucleic acids and one or morebiological assemblers may be assembled, aggregated, commingled, combinedor mixed, or other similar methods. Processes, including sequentialprocesses, are known in the art and include, but are not limited to, oneor more of automated methods, mechanical methods, computer and/orsoftware-controlled methods, and fluid flow. Fluid flow includes, but isnot limited to, nanofluidics and microfluidics. Nanofluidics andmicrofluidics include, but are not limited to, continuous flowmicrofluidics and digital microfluidics, and have been developed for usein biological systems (Annu. Rev. Fluid Mech. (2004) 36:381-411; Annu.Rev. Biomed. Eng. (2002) 4:261-86; Science (1988) 242:1162-1164, Rev.Mod. Phys. (2005) 77:977-1026).

In some embodiments, one or more methods include co-localizing one ormore nucleic acids with one or more biological assemblers at one or morefifth identifiable time intervals. In some embodiments, the one or morefifth identifiable time intervals are at least partially based on apredicted rate of incorporation of amino acids into peptides, apredicted rate of activity of biological assemblers, a predicted rate oftranslocation of nucleic acids and/or a predicted rate of release oftRNA.

In some embodiments, one or more methods that include co-localizing oneor more nucleic acids with one or more biological assemblers at one ormore fifth identifiable time intervals, further include monitoring aminoacid incorporation into peptides, biological assembler activity, nucleicacid translocation, and/or tRNA release. In some embodiments, theresults of monitoring one or more of amino acid incorporation intopeptides, biological assembler activity, nucleic acid translocation, ortRNA release are at least partially used to identify time intervals forco-localization of nucleic acids. In some embodiments, the one or morefifth identifiable time intervals are at least partially based onavailability of one or more nucleic acid codons.

In some embodiments, one or more methods that include co-localizing oneor more nucleic acids with one or more biological assemblers at one ormore fifth identifiable time intervals, further include monitoring thepresence or absence, concentration, and/or compositions of one or morecharged tRNA and/or one or more tRNA. In some embodiments, the resultsof monitoring the presence or absence, concentration, and/orcompositions of one or more charged tRNA and/or one or more tRNA are atleast partially used to identify time intervals for co-localization ofnucleic acids. In some embodiments, the one or more fifth identifiabletime intervals are at least partially based on the presence or absenceof one or more charged tRNA and/or one or more tRNA, and/or the presenceor absence of one or more anti-codons on one or more charged tRNA and/orone or more tRNA.

In some embodiments, one or more methods include co-localizing,optionally sequentially, two or more charged tRNA with one or morebiological assemblers at least partially, or optionally completely,following co-localizing one or more nucleic acids with one or morebiological assemblers. In some embodiments, one or more methods includeco-localizing, optionally sequentially, one or more biologicalassemblers with two or more charged tRNA, at least partially, oroptionally completely, following co-localizing one or more nucleic acidswith the one or more biological assemblers. In some embodiments, one ormore methods include co-localizing, optionally sequentially, one or morebiological assemblers and two or more charged tRNA, at least partially,or optionally completely, following co-localizing one or more nucleicacids with the one or more biological assemblers.

In some embodiments, one or more methods include selecting one or morenucleic acids, including but not limited to, RNA, DNA, cDNA, and mRNA.In some embodiments, the method includes selecting one or more nucleicacids having a selected and/or target protein coding sequence. In someembodiments, one or more nucleic acids are selected based on criteriaincluding, but not limited to, one or more of a target peptide sequence,a target nucleic acid protein coding sequence, one or more charged tRNA,one or more biological assemblers, or one or more biological assemblercomponents. For example, the one or more nucleic acid sequences may beselected at least partially based on the selected target peptidesequence and the selected biological assemblers and/or biologicalassembler components (e.g. Ef-Tu; Biochemistry (2006) 44:11254-11261).In some embodiments, one or more nucleic acids are selected based oncriteria including, but not limited to, user designations, targetoutput, computer predictions, availability, predicted synthetic time,and/or cost.

Some embodiments include any process used to identify for use one ormore target components. Processes include, but are not limited to, userselected, user identified, software method analysis, algorithm-based,computer mediated, operations research, optimization, simulation,queuing theory, and/or game theory.

In some embodiments, one or more of the methods described hereinincludes synthesizing one or more nucleic acids. In some embodiments,one or more nucleic acids are synthesized to have a selected/targetprotein coding sequence. In yet other embodiments, the method includessynthesizing the one or more nucleic acids using two or more singletcodons, two or more doublet codons, two or more triplet codons, two ormore quadruplet codons, two or more quintuplet codons, and/or two ormore sextuplet codons. In other embodiments, the method includessynthesizing one or more nucleic acids using two or more stop codons. Insome embodiments, the method includes synthesizing one or more nucleicacids using two or more different stop codons or three or more differentstop codons. In some embodiments, the nucleic acids are synthesizedhaving alternating stop codons and/or repeating stop codons. In someembodiments, the nucleic acids are synthesized having any one of thetranslatable reading frames described herein.

As used herein, the term “synthesizing” means any process resulting intwo or more nucleotides being joined to form a nucleic acid. Processesto synthesize DNA and RNA are well known to those of skill in the artand include, but are not limited to, one or more of enzymatic orchemical methods such as polymerase chain reaction and phosphoramiditechemistry followed by deprotection, for example.

In some embodiments, one or more methods include synchronizingsequentially providing two or more charged tRNA at one or moreidentifiable locations, with a selected or target protein codingsequence of one or more nucleic acids. In some embodiments, one or moremethods include synchronizing sequentially co-localizing two or morecharged tRNA with the one or more biological assemblers, with a selectedor target protein coding sequence of one or more nucleic acids. In someembodiments, one or more methods include synchronizing sequentiallyco-localizing one or more biological assemblers with two or more chargedtRNA, with a selected or target protein coding sequence of one or morenucleic acids. In some embodiments, one or more methods includesynchronizing sequentially co-localizing one or more biologicalassemblers and two or more charged tRNA, with a selected or targetprotein coding sequence of one or more nucleic acids.

In some embodiments, the nucleic acid protein coding sequence and thetarget protein sequence both follow the standard genetic code, thereforesequential co-localization of charged tRNA also follows the standardgenetic code, and synchronization is based on the standard genetic code.In some embodiments, the target protein coding sequence includes one ormore modified or unnatural amino acids, and synchronization between thetarget peptide sequence and the nucleic acid protein coding sequenceallows the co-localization of a charged tRNA with standard anti-codonrecognition sequence and attached modified/unnatural aminoacyl group. Inother embodiments, the nucleic acid protein coding sequence and targetprotein coding sequence do not follow the standard genetic code,therefore synchronization between the target peptide sequence and thenucleic acid protein coding sequences allows co-localization of chargedtRNA with the anti-codon recognition sequences to pair with the nucleicacid codons and attached aminoacyl groups that follow the target proteincoding sequence.

In other embodiments, one or more of the methods described hereinincludes synchronizing synthesizing one or more nucleic acids, withco-localizing two or more charged tRNA with one or more biologicalassemblers. In other embodiments, one or more of the methods describedherein includes synchronizing synthesizing one or more nucleic acids,with co-localizing one or more biological assemblers with two or morecharged tRNA, wherein at least two of the two or more charged tRNA areat one or more different locations. In other embodiments, one or more ofthe methods described herein includes synchronizing synthesizing one ormore nucleic acids, with sequentially co-localizing one or morebiological assemblers and two or more charged tRNA.

In some embodiments, the nucleic acid protein coding sequence and thetarget protein sequence both follow the standard genetic code, thereforesynchronization of nucleic acid protein coding region synthesis withco-localizing charged tRNA is based on the standard genetic code. Insome embodiments, the target protein coding sequence includes one ormore modified or unnatural amino acids, therefore synchronization allowsa nucleic acid protein coding region to be synthesized with a targetsequence to that coordinates with co-localization (and incorporation) ofa charged tRNA with an attached modified/unnatural aminoacyl group. Inother embodiments, the nucleic acid protein coding sequence and targetprotein coding sequence do not follow the standard genetic code,therefore synchronization of nucleic acid protein coding regionsynthesis with co-localizing charged tRNA allows the synthesis of anucleic acid protein coding sequence with codons that will pair with thecharged tRNA anti-codon recognition sequences having attached aminoacylgroups that follow the target protein coding sequence.

In other embodiments, one or more of the methods described hereinincludes synchronizing synthesizing one or more nucleic acids, andcharging one or more tRNA with one or more natural amino acids, one ormore arbitrary amino acids, and/or one or more unnatural amino acids. Insome embodiments, the nucleic acid protein coding sequence and thetarget protein sequence both follow the standard genetic code, thereforesynchronization of nucleic acid protein coding region synthesis withcharging tRNA with amino acids is based on the standard genetic code. Insome embodiments, the target protein coding sequence includes one ormore modified or unnatural amino acids, therefore synchronization ofnucleic acid protein coding region synthesis and tRNA charging allowsthe coordination of the nucleic acid codon and tRNA anti-codon pairingfor the charged tRNA with an attached modified/unnatural aminoacylgroup. In other embodiments, the nucleic acid protein coding sequenceand target protein coding sequence do not follow the standard geneticcode, therefore synchronization of nucleic acid protein coding regionsynthesis with tRNA charging allows the synthesis of a nucleic acidprotein coding sequence with codons that will pair with the charged tRNAanti-codon recognition sequences having attached aminoacyl groups thatfollow the target protein coding sequence.

As used herein, the term “synchronizing” means any one or more processescoordinating one or more elements of one or more methods. The one ormore elements of one or more methods may include, but are not limited toone or more of two or more processes, or one or more processes and oneor more target sequences. The one or more processes may include, but arenot limited to, user defined, software-based, algorithm-based, computermediated, operations research, optimization, simulation, queuing theory,and/or game theory.

In some embodiments, one or more methods further comprise partially orcompletely isolating the one or more target peptide following synthesis.Methods for isolating proteins are well-known in the art.

In one aspect, the disclosure is drawn to one or more apparatus forpeptide synthesis. In some embodiments, any one of the methods describedherein may be performed on one or more apparatus.

FIG. 1 shows a schematic 400 of an illustrative apparatus 410 forbiologically synthesizing peptides in which embodiments may beimplemented. The apparatus 410 is optionally operable for extra-cellularand/or cell-free peptide synthesis. In some embodiments, the peptidesynthesis is in vitro. The apparatus may optionally be, or include, oneor more units including, but not limited to, one or more peptidesynthesizer units 420, one or more sourcing units 432, one or moremonitoring units 440, one or more controller units 422, one or morecomputing units 426, one or more tRNA charging units 428, and/or one ormore nucleic acid synthesizer units 430. In some embodiments, one ormore of the units may be internal or external to the apparatus.

In some embodiments, one or more apparatus 410 further includes one ormore fluid flows. In some embodiments, the one or more fluid flowsconnect and/or allow the transfer of one or more peptide synthesiscomponents among one or more of the optional one or more units of theapparatus 410. In some embodiments, the one or more fluid flows areoperable to provide, co-localize, remove and/or separate, optionallysequentially, one or more peptide synthesis components. In someembodiments, the one or more fluid flows are operable to provide,co-localize, remove and/or separate, optionally sequentially, one ormore peptide synthesis components at one or more identifiable timeintervals.

FIG. 2 shows a schematic 400 of illustrative embodiments of the optionalapparatus 410 of FIG. 1, with specific illustrative embodiments of oneor more peptide synthesizer units 420, including unit 4200, unit 4201,unit 4202, and unit 4203. In some embodiments, one or more peptidesynthesizer units are operable to provide, optionally sequentially, oneor more peptide synthesis components to one or more identifiablelocations. In some embodiments, one or more peptide synthesizer unitsare operable to co-localize, optionally sequentially, one or morepeptide synthesis components. In some embodiments, one or more peptidesynthesizer units are operable to remove, optionally sequentially, oneor more peptide synthesis components from one or more identifiablelocations. In some embodiments, one or more peptide synthesizer unitsare operable to separate, optionally sequentially, one or more peptidesynthesis components.

In some embodiments, the one or more peptide synthesizer units areoperable to provide, co-localize, remove, and/or separate, optionallysequentially, one or more peptide synthesis components at one or morefirst identifiable locations, wherein the one or more identifiablelocations include one more temporal-spatial locations. In someembodiments, one or more temporal-spatial locations are moving alongpredictable time or other sequential path. In some embodiments, the oneor more identifiable locations are one location. In some embodiments,the one or more identifiable locations are external to the apparatus. Insome embodiments, the one or more identifiable locations are internal tothe apparatus, and/or one or more of the optional units within theapparatus. In some embodiments, each operable element may have adifferent identifiable location or one or more operable elements mayhave similar and/or identical identifiable locations.

In some embodiments, the one or more peptide synthesizer units areoperable to provide, co-localize, remove, and/or separate, optionallysequentially, one or more peptide synthesis components at one or moreidentifiable time intervals. In some embodiments, the one or more firstidentifiable time intervals are at least partially based on a predictedrate of incorporation of two or more amino acids into one or morepeptides, a predicted rate of activity of one or more biologicalassemblers, a predicted rate of translocation of one or more nucleicacids, and/or a predicted rate of release of tRNA. In some embodiments,the one or more first identifiable time intervals are from approximately0.001 seconds to approximately 0.01 seconds, or are approximately 0.01seconds, and/or other appropriate time intervals as described elsewhere.In some embodiments, each operable element may have a differentidentifiable time interval or one or more operable elements may have asimilar and/or identical identifiable time interval.

In some embodiments, the one or more peptide synthesizer units includeone or more fluid flows. The one or more fluid flows may be used toprovide and/or co-localize components for peptide synthesis in one ormore identifiable locations. The one or more fluid flows may be used toremove and/or separate components for peptide synthesis from the one ormore identifiable locations. The one or more fluid flows may be used totransfer one or more charged tRNA, one or more biological assemblers,one or more biological assembler components, and/or one or more nucleicacids to and/or from one or more identifiable locations. In someembodiments, the one or more fluid flows are operable to transfer one ormore peptide synthesis components to one or more identifiable locationsat one or more identifiable time intervals.

In some embodiments, the one or more peptide synthesizer units areoperable to optionally affix one or more peptide synthesis components,including but not limited to, one or more biological assemblers, one ormore charged tRNA, one or more nucleic acids, and/or one or morebiological assembler components. In some embodiments, one or morepeptide synthesis components are affixed at one or more identifiablelocations.

In some embodiments, one or more peptide synthesizer units areoptionally operable to isolate one or more target peptides followingpartial complete synthesis.

In one aspect, the disclosure is drawn to one or more apparatuscomprising one or more peptide synthesizer units that are operable toco-localize two or more charged tRNA with one or more biologicalassemblers at one or more first identifiable locations. Unit 4200 isoptionally one or more peptide synthesizer units operable to provideand/or to remove one or more charged tRNA and/or released tRNA to and/orfrom one or more first identifiable locations. In some embodiments, oneor more peptide synthesizer units are operable to sequentially providetwo or more charged tRNA to one or more first identifiable locations. Insome embodiments, one or more peptide synthesizer units are furtheroperable to remove and/or separate, optionally sequentially, two or morecharged tRNA and/or tRNA and/or other components from one or more firstidentifiable locations. In some embodiments, one or more peptidesynthesizer units are operable to provide, optionally sequentially, twoor more charged tRNA to one or more first identifiable locations, and toremove and/or separate, optionally sequentially, two or more chargedtRNA and/or one or more tRNA from one or more first identifiablelocations.

In some embodiments, the one or more peptide synthesizer units areoperable to optionally sequentially provide two or more charged tRNA toone or more identifiable locations at one or more first identifiabletime intervals. In some embodiments, the one or more peptide synthesizerunits are further operable to optionally sequentially separate two ormore charged tRNA from one or more identifiable locations at one or moresecond identifiable time intervals.

Unit 4201 is optionally one or more peptide synthesizer units operableto provide and/or to remove one or more biological assemblers to and/orfrom one or more identifiable locations. In some embodiments, one ormore apparatus include one or more peptide synthesizer units that areoperable to provide, optionally sequentially, one or more biologicalassemblers to one or more first identifiable locations. In someembodiments, one or more units are further operable to remove,optionally sequentially, one or more biological assemblers from one ormore first identifiable locations.

In some embodiments, the one or more peptide synthesizer units arefurther operable to affix one or more biological assemblers at one ormore first identifiable locations. In some embodiments, the one or morebiological assemblers are affixed at one or more first identifiablelocations.

In some embodiments, the one or more peptide synthesizer units areoperable to co-localize, optionally sequentially, one or more biologicalassemblers with two or more charged tRNA, wherein at least two of thetwo or more charged tRNA are at one or more different locations. In someembodiments, one or more units are further operable to separate,optionally sequentially, the one or more biological assemblers from thetwo or more charged tRNA. In some embodiments, one or more apparatusinclude one or more peptide synthesizer units that are operable toco-localize, optionally sequentially, one or more biological assemblersand two or more charged tRNA, at one or more identifiable locations. Insome embodiments, one or more units are further operable to separate,optionally sequentially, the one or more biological assemblers and thetwo or more charged tRNA.

In some embodiments, the one or more biological assemblers may be one ormore peptide assemblers, one or more ribosome-based biologicalassemblers, and/or one or more non-ribosome-based biological assemblers.The one or more ribosome-based biological assemblers may be eukaryotic,mitochondrial and/or prokaryotic, among others.

Unit 4202 is optionally one or more peptide synthesizer units operableto provide and/or to remove one or more biological assembler componentsto and/or from one or more identifiable locations. In some embodiments,one or more apparatus include one or more peptide synthesizer units thatare operable to provide, optionally sequentially, one or more biologicalassemblers components to one or more second identifiable locations. Insome embodiments, one or more peptide synthesizer units are operable toremove, optionally sequentially, one or more biological assemblercomponents from one or more second identifiable locations. The one ormore second identifiable locations are optionally the same as, oroptionally different from the first one or more identifiable locations.

The one or more biological assembler components, may be one or morepeptide assembler components, one or more ribosome-based biologicalassembler components, and/or one or more non-ribosome-based biologicalassembler components. The one or more ribosome-based biologicalassembler components may be eukaryotic, mitochondrial and/orprokaryotic, among others.

Unit 4203 is optionally one or more peptide synthesizer units operableto provide and/or to remove one or more nucleic acids to and/or from oneor more identifiable locations. In some embodiments, one or moreapparatus include one or more peptide synthesizer units that areoperable to provide, optionally sequentially, one or more nucleic acidsto one or more first identifiable locations. In some embodiments, one ormore apparatus includes one or more peptide synthesizer units that areoperable to remove, optionally sequentially, one or more nucleic acidsfrom one or more first identifiable locations. In some embodiments, oneor more nucleic acids are one or more DNA, one of more cDNA, one or moreRNA and/or one or more mRNA.

FIG. 3 shows a schematic 400 of illustrative embodiments of theapparatus 410 of FIG. 1, with specific illustrative embodiments of oneor more sourcing units 432, including unit 4320, unit 4322, unit 4321,unit 4323, unit 4324, unit 4325, unit 4326, unit 4327, and/or unit 4328.In some embodiments, one or more sourcing units 432 optionally containone or more peptide synthesis components. In some embodiments, one ormore apparatus includes, but is not limited to, one or more peptidesynthesizer units 420 and one or more sourcing units 432. In someembodiments, one or more of the one or more peptide synthesizer units420 and one or more of the one or more sourcing units 432 are the sameunit. In some embodiments, one or more sourcing units 432 include one ormore fluid flows. In some embodiments, one or more sourcing units 432are operable to provide/co-localize/remove/separate one or more peptidesynthesis components from one or more identifiable locations.

In some embodiments, one or more sourcing units 432 are operable toprovide/co-localize/remove/separate one or more peptide synthesiscomponents from one or more identifiable locations at one or moreidentifiable time intervals. In some embodiments, the one or moreidentifiable time intervals are at least partially based on a predictedrate of incorporation of two or more amino acids into one or morepeptides, a predicted rate of activity of one or more biologicalassemblers, a predicted rate of translocation of one or more nucleicacids, and/or a predicted rate of release of tRNA. In some embodiments,the one or more identifiable time intervals are from approximately 0.001seconds to approximately 0.1 seconds and/or approximately 0.01 seconds,or other appropriate time interval.

In some embodiments, one or more sourcing units include one or moresources of charged tRNA 4320, one or more sources of biologicalassemblers 4322, one or more sources of biological assembler components4323, one or more sources the nucleic acids 4321, one or more sources ofDNA, one or more sources of cDNA, one or more sources of mRNA, one ormore sources of RNA, one or more sources of tRNA 4324, one or moresources of amino acids 4326 one or more sources of nucleotides 4327, oneor more sources of tRNA charging components 4325, and/or one or moresources of nucleic acid synthesis components 4328. In some embodiments,one or more sourcing units 432 include one or more of one or moresources of tRNA 4324 (including, but not limited to, natural, unnatural,and arbitrary tRNA), one or more sources of amino acids 4326 (including,but not limited to, natural, unnatural, and arbitrary amino acids),and/or one or more sources of tRNA charging components 4325 (including,but not limited to, natural, unnatural, and arbitrary).

Unit 4320 is optionally one or more sourcing units containing one ormore sources of charged tRNA, and is optionally operable to provideand/or to remove one or more charged tRNA and/or released tRNA to and/orfrom one or more identifiable locations. In some embodiments, one ormore apparatus includes one or more or two or more sources of chargedtRNA 4320 optionally operable to provide two or more charged tRNA to oneor more identifiable locations at one or more first identifiable timeintervals, and/or to remove one or more charged tRNA and/or releasedtRNA from one or more identifiable locations at one or more secondidentifiable time intervals.

In some embodiments, one or more apparatus includes two or more sourcesof charged tRNA 4320, wherein one or more first source of the two ormore sources of charged tRNA includes a supply of one or more first typeof charged tRNA, and one or more second source of the two or moresources of charged tRNA includes one or more second type of chargedtRNA. In some embodiments, the one or more first type of charged tRNA isdifferent from, or the same as, the one or more second type of chargedtRNA. In some embodiments, one or more apparatus further includes one ormore third source of the two or more sources of charged tRNA thatincludes a supply of one or more third type of charged tRNA. In someembodiments, the one or more third type of charged tRNA is differentfrom, or the same as, the one or more first type of charged tRNA and/orthe one or more second type of charged tRNA. In some embodiments, thefirst type of charged tRNA includes one or more natural charged tRNA,one or more unnatural charged tRNA, and/or one or more arbitrary chargedtRNA and the second type of charged tRNA includes one or more naturalcharged tRNA, one or more unnatural charged tRNA, and/or one or morearbitrary charged tRNA. In some embodiments, the two or more sources ofcharged tRNA include one or more fluid flows.

Unit 4322 is optionally one or more sourcing units containing one ormore sources of biological assemblers, and is optionally operable toprovide and/or to remove one or more biological assemblers to and/orfrom one or more identifiable locations. In some embodiments, one ormore apparatus includes one or more sources of biological assemblers4322 optionally operable to provide and/or to remove one or morebiological assemblers to and/or from one or more identifiable locationsat one or more third identifiable time intervals. In some embodiments,one or more apparatus includes one or more sources of biologicalassemblers 4322, wherein one or more first source of the one or morebiological assemblers includes a supply of one or more first type ofbiological assemblers, and one or more second source of the one or moresources of biological assemblers includes one or more second type ofbiological assemblers. In some embodiments, the one or more first typeof biological assemblers is different from, or the same as, the one ormore second type of biological assemblers. In some embodiments, one ormore apparatus further includes one or more third source of the one ormore sources of biological assemblers that includes a supply of one ormore third type of biological assemblers. In some embodiments, the oneor more third type of biological assemblers is different from, or thesame as, the one or more first type of biological assemblers and/or theone or more second type of biological assemblers. In some embodiments,the first type of biological assemblers is prokaryotic, and the secondtype of biological assemblers in eukaryotic. In some embodiments, theone or more sources of biological assemblers include one or more fluidflows.

Unit 4321 is optionally one or more sourcing units containing one ormore sources of nucleic acids, and is optionally operable to provideand/or to remove one or more nucleic acids to and/or from one or moreidentifiable locations. In some embodiments, one or more apparatusincludes one or more sources of nucleic acids 4321 optionally operableto provide and/or to remove one or more nucleic acids to and/or from oneor more identifiable locations at one or more fourth identifiable timeintervals. In some embodiments, one or more apparatus includes one ormore sources of nucleic acids 4321, wherein one or more first source ofthe one or more nucleic acids includes a supply of one or more firsttype of nucleic acids, and one or more second source of the one or moresources of nucleic acids includes one or more second type of nucleicacids. In some embodiments, the one or more first type of nucleic acidsis different from, or the same as, the one or more second type ofnucleic acids. In some embodiments, one or more apparatus furtherincludes one or more third source of the one or more sources of nucleicacids that includes a supply of one or more third type of nucleic acids.In some embodiments, the one or more third type of nucleic acids isdifferent from, or the same as, the one or more first type of nucleicacids and/or the one or more second type of nucleic acids. In someembodiments, the first type of nucleic acids is DNA and the second typeof nucleic acids is RNA. In some embodiments, the one or more sources ofnucleic acids are one or more sources of RNA, one or more sources ofmRNA, one or more sources of DNA, and/or one or more sources of cDNA. Insome embodiments, the one or more sources of nucleic acids include oneor more fluid flows. In some embodiments, the one or more thirdlocations are included in the two or more first locations and/or areoptionally the same as one or more second locations.

Unit 4323 is optionally one or more sourcing units containing one ormore sources of biological assembler components, and is optionallyoperable to provide and/or to remove one or more biological assemblercomponents to and/or from one or more identifiable locations. In someembodiments, one or more apparatus includes one or more sources ofbiological assembler components 4323, each source positioned to provideone or more biological assembler components to one or more fourthlocations. In some embodiments, one or more of the one or more sourcesof biological assembler components includes one or more fluid flows. Insome embodiments, the one or more fourth locations are one or moretemporal-spatial locations and/or are moving along a predictable time orother sequential path. In some embodiments, the one or more fourthlocations are optionally the same as the one or more sources of one ormore biological assemblers and/or the one or more first locations. Insome embodiments, the one or more sources of biological assemblercomponents are positioned to provide the biological assembler componentsto one or more sources of one or more biological assemblers and/or theone or more first locations.

In some embodiments, one or more apparatus includes one or more sourcesof biological assembler components 4323, wherein one or more firstsource of the one or more biological assembler components includes asupply of one or more first type of biological assembler components, andone or more second source of the one or more sources of biologicalassembler components includes one or more second type of biologicalassembler components. In some embodiments, the one or more first type ofbiological assembler components is different from, or the same as, theone or more second type of biological assembler components. In someembodiments, one or more apparatus further includes one or more thirdsource of the one or more sources of biological assembler componentsthat includes a supply of one or more third type of biological assemblercomponents. In some embodiments, the one or more third type ofbiological assembler components is different from, or the same as, theone or more first type of biological assembler components and/or the oneor more second type of biological assembler components. In someembodiments, the one or more sources of biological assembler componentsinclude one or more fluid flows.

In some embodiments, one or more apparatus includes one or more sourcesof tRNA 4324, each source positioned to provide one or more tRNA to oneor more second locations; one or more sources of amino acids 4326, eachsource positioned to provide one or more amino acids to the one or moresecond locations; and one or more sources of tRNA charging components4325, each source positioned to provide one or more tRNA chargingcomponents to the one or more second locations. In some embodiments, oneor more of the one or more sources of tRNA, the one or more sources ofamino acids, and/or the one or more sources of tRNA charging componentsinclude one or more fluid flows. In some embodiments, the one or moresecond locations are one or more temporal-spatial locations and/or theone or more temporal-spatial locations are moving along a predictabletime or other sequential path. In some embodiments, the one or moresecond locations are positioned to provide charged tRNA to the two ormore sources of charged tRNA. In some embodiments, the one or moresecond locations are one or more sources of charged tRNA.

Unit 4324 is optionally one or more sourcing units containing one ormore sources of tRNA, and is optionally operable to provide and/or toremove one or more tRNA to and/or from one or more identifiablelocations. In some embodiments, one or more first source of the one ormore sources of tRNA includes a supply of one or more first type of tRNAand one or more second source of the one or more sources of tRNAincludes a supply of one or more second type of tRNA. In someembodiments, the one or more first type of tRNA is different from, orthe same as, the one or more second type of tRNA. In some embodiments,the one or more first type of tRNA includes one or more natural tRNA,and the one or more second type of tRNA includes one or more unnaturaltRNA.

Unit 4326 is optionally one or more sourcing units containing one ormore sources of amino acids, and is optionally operable to provideand/or to remove one or more amino acids to and/or from one or moreidentifiable locations. In some embodiments, one or more first source ofthe one or more sources of amino acids includes a supply of one or morefirst type of amino acids and one or more second source of the one ormore sources of amino acids includes a supply of one or more second typeof amino acids. In some embodiments, the one or more first type of aminoacids is different from, or the same as, the one or more second type ofamino acids. In some embodiments, the one or more first type of aminoacids includes one or more natural amino acids, and the one or moresecond type of amino acids includes one or more unnatural amino acids.

Unit 4325 is optionally one or more sourcing units containing one ormore sources of tRNA charging components, and is optionally operable toprovide and/or to remove one or more tRNA charging components to and/orfrom one or more identifiable locations. In some embodiments, one ormore first source of the one or more sources of tRNA charging componentsincludes a supply of one or more first type of tRNA charging components,and one or more second source of the one or more sources of tRNAcharging components includes a supply of one or more second type of tRNAcharging components. In some embodiments, the one or more first type oftRNA charging components is different from, or the same as, the one ormore second type of tRNA charging components. In some embodiments, theone or more first type of tRNA charging components include one or moretRNA synthetases, and the one or more second type of tRNA chargingcomponents includes one or more non-natural tRNA charging components. Insome embodiments, the first type of tRNA charging components include oneor more prokaryotic tRNA synthetases, and the second type of tRNAcharging components include one or more eukaryotic tRNA synthetases.

Unit 4327 is optionally one or more sourcing units containing one ormore sources of nucleotides, and is optionally operable to provideand/or to remove one or more nucleotides to and/or from one or moreidentifiable locations. Unit 4328 is optionally one or more sourcingunits containing one or more sources of nucleic acid synthesiscomponents, and is optionally operable to provide and/or to remove oneor more nucleic acid synthesis components to and/or from one or moreidentifiable locations. In some embodiments, one or more apparatusincludes one or more sources of nucleotides 4327, each source positionedto provide one or more nucleotides to one or more third locations, andone or more sources of nucleic acid synthesis components 4328, eachsource positioned to provide one or more nucleic acid synthesiscomponents to the one or more third locations. In some embodiments, oneor more of the one or more sources of nucleotides, each sourcepositioned to provide one or more nucleotides to one or more thirdlocations, and one or more sources of nucleic acid synthesis components,each source positioned to provide one or more nucleic acid synthesiscomponents to the one or more third locations include fluid flows. Insome embodiments, the one or more third locations is one or moretemporal-spatial locations and/or the one or more temporal-spatiallocations are moving along a predictable time or other sequential path.In some embodiments, the one or more third locations are positioned toprovide nucleic acids to one or more sources of nucleic acids. In someembodiments, the one or more third locations are one or more sources ofnucleic acids 4321.

In one aspect, the disclosure is drawn to one or more apparatuscomprising two or more sources of charged tRNA, each source positionedto sequentially provide one or more charged tRNA to one or more firstlocations. In some embodiments, one or more apparatus includes two ormore sources of charged tRNA 4320, each source positioned tosequentially provide one or more charged tRNA to one or more firstlocations, and optionally to remove one or more charged tRNA and/or oneor more tRNA from one or more first locations. In some embodiments, oneor more apparatus includes two or more sources of charged tRNA 4320,each source positioned to sequentially provide one or more charged tRNAto one or more first locations containing one or more biologicalassemblers, and optionally to remove one or more charged tRNA and/or oneor more tRNA from one or more first locations containing one or morebiological assemblers.

In some embodiments, one or more apparatus includes two or more sourcesof charged tRNA 4320, each source positioned to sequentially provide oneor more charged tRNA to one or more first locations and optionally toremove one or more charged tRNA and/or one or more tRNA from one or morefirst locations; and one or more sources of biological assemblers 4322.In some embodiments, one or more apparatus includes two or more sourcesof charged tRNA 4320, each source positioned to sequentially provide oneor more charged tRNA to one or more first locations and optionally toremove one or more charged tRNA and/or one or more tRNA from one or morefirst locations; and one or more sources of biological assemblers 4322,each source positioned to provide, and optionally to remove, one or morebiological assemblers to/from the one or more first locations. In someembodiments, the one or more biological assemblers are affixed.

In some embodiments, one or more apparatus includes two or more sourcesof charged tRNA 4320, each source positioned to sequentially provide oneor more charged tRNA to one or more first locations at one or more firstidentifiable time intervals, and optionally to remove one or morecharged tRNA and/or one or more tRNA from one or more first locations atone or more second identifiable time intervals. In some embodiments, oneor more apparatus includes two or more sources of charged tRNA 4320,each source positioned to sequentially co-localize one or more chargedtRNA with one or more first locations containing one or more biologicalassemblers at one or more first identifiable time intervals, andoptionally to remove and/or separate one or more charged tRNA and/or oneor more tRNA from one or more first locations containing one or morebiological assemblers at one or more second identifiable time intervals.

In some embodiments, one or more apparatus includes two or more sourcesof charged tRNA 4320, each source positioned to sequentially provide oneor more charged tRNA to one or more first locations at one or more firstidentifiable time intervals, and optionally to remove one or morecharged tRNA and/or one or more tRNA from one or more first locations atone or more second identifiable time intervals; and one or more sourcesof biological assemblers 4322. In some embodiments, one or moreapparatus includes two or more sources of charged tRNA 4320, each sourcepositioned to sequentially provide one or more charged tRNA to one ormore first locations at one or more first identifiable time intervals,and optionally to remove one or more charged tRNA and/or one or moretRNA from one or more first locations at one or more second identifiabletime intervals; and one or more sources of biological assemblers 4322,each source positioned to provide, and optionally to remove, one or morebiological assemblers to/from the one or more first locations at one ormore third identifiable time intervals. In some embodiments, the one ormore biological assemblers are affixed.

In some embodiments, one or more apparatus includes two or more sourcesof charged tRNA, each source further positioned to provide, optionallysequentially, two or more charged tRNA to one or more first locations,and further includes one or more fluid flows. In some embodiments, theone or more fluid flows provide, optionally sequentially, the two ormore charged tRNA to the one or more locations, and optionally remove,optionally sequentially, one or more charged tRNA and/or one or moretRNA from the one or more first locations. In some embodiments, the oneor more fluid flows provide, optionally sequentially, the one or morebiological assemblers to the one or more locations, and optionallyremove, optionally sequentially, the one or more biological assemblersfrom the one or more first locations.

In one aspect, the disclosure is drawn to one or more apparatuscomprising two or more sources of charged tRNA; and one or more sourcesof biological assemblers, each source positioned to sequentially provideone or more biological assemblers to two or more first locations, and tooptionally remove, optionally sequentially, the one or more biologicalassemblers from the two or more first locations. In some embodiments,one or more apparatus includes two or more sources of charged tRNA 4320,each source positioned to provide, optionally sequentially, one or morecharged tRNA to one or more second locations, and optionally to remove,optionally sequentially, one or more charged tRNA and/or one or moretRNA from one or more second locations; and one or more sources ofbiological assemblers 4322, each source positioned to sequentiallyprovide one or more biological assemblers to two or more first locationsand optionally to remove, optionally sequentially, the one or morebiological assemblers from the two or more first locations. In someembodiments, one or more apparatus includes two or more sources ofcharged tRNA 4320, each source positioned to affix, optionallysequentially, one or more charged tRNA at one or more second locations.In some embodiments, one or more of the two or more charged tRNA areaffixed optionally at the one or more second locations.

In some embodiments, one or more apparatus includes one or more sourcesof biological assemblers 4322 and/or two or more sources of charged tRNA4320 each including one or more fluid flows. In some embodiments, one ormore apparatus further includes one or more fluid flows, wherein one ormore of the one or more fluid flows optionally sequentially provides theone or more biological assemblers to each of the two or more firstlocations and optionally sequentially removes the one or more biologicalassemblers from each of the two or more first locations. In someembodiments, one or more apparatus further includes one or more fluidflows, wherein one or more of the one or more fluid flows optionallysequentially provides the one or more charged tRNA to each of the two ormore second locations and optionally sequentially removes the one ormore charged tRNA and/or tRNA from each of the two or more secondlocations.

In some embodiments, one or more apparatus includes two or more sourcesof charged tRNA; and one or more sources of biological assemblers, eachsource positioned to sequentially provide one or more biologicalassemblers to two or more first locations at one or more firstidentifiable time intervals, and to optionally remove, optionallysequentially, the one or more biological assemblers from the two or morefirst locations at one or more second identifiable time intervals. Insome embodiments, one or more apparatus further include two or moresources of charged tRNA 4320, each source positioned to provide one ormore charged tRNA to one or more second locations at one or more thirdidentifiable time intervals, and optionally to remove one or morecharged tRNA from one or more second locations at one or more fourthidentifiable time intervals.

In some embodiments, the first locations and/or second locations are oneor more temporal-spatial locations and/or are moving along a predictabletime or other sequential path. In some embodiments, the two or morefirst locations are the two or more sources of charged tRNA. In someembodiments, one or more of the one or more second locations are one ormore of the one or more first locations.

FIG. 4 shows a schematic 400 of illustrative embodiments of theapparatus 410 of FIG. 1, with specific illustrative embodiments of oneor more monitoring units 440, including unit 4400 and unit 4401. In someembodiments, one or more apparatus includes, but is not limited to, oneor more peptide synthesizer units 420 and one or more monitoring units440. In some embodiments, the one or more peptide synthesizer units 420and the one or more monitoring units 440 are the same one or more units.In some embodiments, one or more apparatus includes, but is not limitedto one or more peptide synthesizer units 420, one or more sourcing units432, and one or more monitoring units 440. In some embodiments, the oneor more peptide synthesizer units 420, one or more sourcing units 432,and the one or more monitoring units 440 are the same unit.

Unit 4400 is optionally one or more monitoring units operable to measureone or more elements of peptide synthesis. Specific illustrativeembodiments of unit 4400, include but are not limited to, one or moremonitoring units operable to measure amino acid incorporation 44000, oneor more monitoring units operable to measure biological assembleractivity 44001, one or more monitoring units operable to measure nucleicacid translocation 44002, and/or one or more monitoring units operableto measure tRNA release 44003.

Unit 4401 is optionally one or more monitoring units, operable tomeasure presence and/or absence, concentration, and/or composition ofone or more peptide synthesis components. Specific illustrativeembodiments of unit 4401 include, but are not limited to, unit 44010,unit 44011, unit 44012, unit 44013, and/or 44014. Unit 44010 isoptionally one or more monitoring units operable to measure presenceand/or absence, concentration, and/or composition of one or more of thetwo or more charged tRNA. Unit 44011 is optionally one or moremonitoring units operable to measure presence and/or absence,concentration, and/or composition of one or more tRNA. Unit 44012 isoptionally one or more monitoring units operable to measure presenceand/or absence, concentration, and/or composition of one or morebiological assemblers. Unit 44013 is optionally one or more monitoringunits operable to measure presence and/or absence, concentration, and/orcomposition of one or more biological assembler components. Unit 44014is optionally one or more monitoring units operable to measure presenceand/or absence, concentration, and/or composition of one or more nucleicacids.

FIG. 5 shows a schematic 400 of illustrative embodiments of theapparatus 410 of FIG. 1, with specific illustrative embodiments of oneor more controller units 422, including unit 4220, unit 4221, unit 4222,unit 4223, unit 4224, and unit 4225, wherein one or more of these unitsare optionally the same unit. In some embodiments, one or more apparatusincludes, but is not limited to, one or more peptide synthesizer units420 and one or more controller units 422. In some embodiments, the oneor more peptide synthesizer units 420 and the one or more controllerunits 422 are the same one or more units. In some embodiments, one ormore apparatus includes, but is not limited to one or more peptidesynthesizer units 420, one or more sourcing units 432, one or morecontroller units 422 and one or more monitoring units 440. In someembodiments, the one or more peptide synthesizer units 420, one or moresourcing units 432, the one or more controller units 422 and the one ormore monitoring units 440 are the same unit. In some embodiments, theone or more controller units 422 control the activity of the one or moreunits of one or more apparatus 410.

In some embodiments, one or more controller units 422 are operable tocontrol the timing and/or the order for providing and/or co-localizingone or more peptide synthesis components at one or more identifiablelocations, and optionally further operable to control the timing and/orthe order for removing and/or separating one or more peptide synthesiscomponents from one or more identifiable locations. The one or morepeptide synthesis components include, but are not limited to, one ormore charged tRNA, one or more tRNA, one or more biological assemblers,one or more biological assembler components, one or more nucleic acidsand/or one or more nucleic acid synthesizing components.

In some embodiments, one or more controller units 422 are furtheroperable to control an order and/or timing for providing and/orco-localizing one or more charged tRNA assembly components at one ormore identifiable locations, and optionally further operable to controlthe timing and/or the order for removing and/or separating one or morecharged tRNA assembly components from one or more identifiablelocations. The one or more charged tRNA assembly components include, butare not limited to, one or more tRNA, one or more amino acids, and/orone or more tRNA charging components.

In some embodiments, one or more controller units 422 are furtheroperable to control an order and/or timing for providing and/orco-localizing one or more nucleic acid assembly components at one ormore identifiable locations, and optionally further operable to controlthe timing and/or the order for removing and/or separating one or morenucleic acid assembly components from one or more identifiablelocations. The one or more nucleic acid assembly components include, butare not limited to, one or more nucleotides, and/or one or more nucleicacid synthesis components.

In some embodiments, the timing is at least partially based on themechanism of peptide synthesis performed by the apparatus. Mechanisms ofpeptide synthesis include, but are not limited to, charged tRNA beingprovided sequentially to one or more locations, charged tRNA beingprovided sequentially to affixed biological assemblers; biologicalassemblers being provided sequentially to affixed charged tRNA; and/orbiological assemblers and charged tRNA being co-localized at one or morelocations. Depending on the mechanism of peptide synthesis, the timingrelated to providing/removing charged tRNA and/or the timing related toproviding/removing biological assemblers, for example, optionallychanges. Timing for each element may be different, and may changedepending on the mechanism of synthesis and/or the biological synthesiscomponents.

In some embodiments, the timing includes, but is not limited to,sequential timing, fixed timing, variable timing, predicted timing, anddata-driven timing. In some embodiments, the timing is one or moreidentifiable time intervals. In some embodiments, the one or moreidentifiable time intervals are from approximately 0.001 seconds to 0.1seconds and/or approximately 0.01 seconds, or other appropriate timeintervals described elsewhere.

In some embodiments, the order and/or the timing for providing,co-localizing, removing and/or separating one or more peptide synthesiscomponents is at least partially based on a target peptide sequenceand/or a nucleic acid protein coding sequence. In some embodiments, theorder and/or the timing for providing, co-localizing, removing and/orseparating one or more peptide synthesis components is at leastpartially based on a predicted rate of incorporation of two or moreamino acids into one or more peptides, a predicted rate of activity ofone or more biological assemblers, a predicted rate of translocation ofone or more nucleic acids, and/or a predicted rate of release of tRNA.

In some embodiments, one or more controller units is operable to controlthe order and/or the timing for providing, co-localizing, removingand/or separating one or more peptide synthesis components at leastpartially based on monitoring of amino acid incorporation into one ormore peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. In some embodiments, one or moremonitoring units 440 are operable to perform the monitoring of aminoacid incorporation into one or more peptides, biological assembleractivity, nucleic acid translocation, and/or tRNA release. In someembodiments, one or more of the one or more monitoring units 440 and oneor more of the one or more controller units 422 are the same units.

In some embodiments, one or more controller units 422 is operable tocontrol the order and/or the timing for providing, co-localizing,removing and/or separating one or more peptide synthesis components atleast partially based on measurements including, but not limited to,availability of one or more nucleic acid codons, concentrations of oneor more of the two or more charged tRNA or the one or more tRNA,presence or absence of one or more of the two or more charged tRNA orthe one or more tRNA, or presence or absence of one or more anti-codonson one or more of the two or more charged tRNA or the one or more tRNA.In some embodiments, one or more of these measurements is at leastpartially determined extrinsically. In some embodiments, one or more ofthese measurements are determined based at least partially onmeasurements by one or more monitoring units 440. In some embodiments,one or more of these measurements are provided in real time.

In illustrative embodiments, one or more controller units are operableto provide (and optionally to remove) two or more charged tRNA in asequence at one or more identifiable time intervals to one or morebiological assemblers at one or more identifiable locations, and arefurther operable to co-localize (and optionally to remove) at one ormore identifiable time intervals the one or more biological assemblersat the one or more identifiable locations. The one or more identifiabletime intervals are optionally different for the co-localization of thetwo or more charged tRNA, the co-localization of one or more biologicalassemblers, the removal of one or more charged tRNA and/or one or moretRNA, and/or the removal of one or more biological assemblers.

In illustrative embodiments, one or more controller units are operableto co-localize (and optionally to separate) one or more biologicalreceptors with two or more charged tRNA in a sequence at one or moreidentifiable time intervals, wherein at least two of the two or morecharged tRNA are at one or more different locations. The one or moreidentifiable time intervals are optionally different for theco-localization of the one or more biological assemblers with the two ormore charged tRNA, the separation of the one or more biologicalassemblers from the two or more charged tRNA, the co-localization of thetwo or more charged tRNA at the one or more different locations and/orthe removal of the two or more charged tRNA from the one or moredifferent locations.

In illustrative embodiments, one or more controller units are operableto co-localize (and optionally to separate) two or more charged tRNA andone or more biological assemblers in a sequence at one or moreidentifiable locations and at one or more identifiable time intervals.The one or more identifiable time intervals are optionally different forthe co-localization of two or more charged tRNA, the co-localization ofone or more biological assemblers, the removal of one or more chargedtRNA and/or one or more tRNA, and/or the removal of one or morebiological assemblers.

Unit 4220 is optionally one or more controller units, operable tocontrol the timing and/or order for providing and/or removing one ormore or two or more charged tRNA and/or released tRNA to and/or from oneor more identifiable locations. In some embodiments, one or moreapparatus include, but are not limited to, one or more peptidesynthesizer units and one or more controller units that are operable tocontrol the timing for providing and optionally for removing one or morecharged tRNA and/or one or more tRNA. In some embodiments, one or moreof the one or more peptide synthesizer units and one or more of the oneor more charged tRNA controller units are the same unit.

In some embodiments, one or more controller units are operable tocontrol the timing and/or the order for providing, optionallysequentially, two or more charged tRNA to one or more first identifiablelocations, and optionally further operable to control the timing and/orthe order for removing, optionally sequentially, two or more chargedtRNA and/or one or more tRNA from one or more first identifiablelocations. In some embodiments, one or more controller units areoperable to control the timing and/or order for sequentiallyco-localizing two or more charged tRNA with one or more biologicalassemblers, and optionally sequentially removing two or more chargedtRNA and/or one or more tRNA from the one or more biological assemblers.In some embodiments, one or more controller units are operable tocontrol the timing and/or order for sequentially co-localizing two ormore charged tRNA and one or more biological assemblers, and optionallysequentially separating the two or more charged tRNA and/or one or moretRNA and the one or more biological assemblers.

Unit 4221 is optionally one or more controller units, operable tocontrol the timing and/or order for providing and/or removing,optionally sequentially, one or more biological assemblers to and/orfrom one or more identifiable locations. In some embodiments, one ormore apparatus include, but are not limited to, one or more peptidesynthesizer units and one or more controller units that are operable tocontrol the timing for providing and optionally for removing one or morebiological assemblers. In some embodiments, one or more of the one ormore peptide synthesizer units and one or more of the one or morebiological assembler controller units are the same unit.

In some embodiments, one or more controller units are operable tocontrol the timing and/or order for, optionally sequentially,co-localizing one or more biological assemblers, and optionally furtheroperable to control the timing and/or order for, optionallysequentially, removing the one or more biological assemblers followingpeptide synthesis. In some embodiments, one or more controller units areoperable to control the timing and/or order for, optionallysequentially, co-localizing one or more biological assemblers with twoor more charged tRNA, wherein at least two of the two or more chargedtRNA are at one or more different locations, and optionally furtheroperable to control the timing and/or order for, optionallysequentially, separating the one or more biological assemblers from thetwo or more charged tRNA and/or one or more tRNA. In some embodiments,one or more controller units are operable to control the timing and/ororder for, optionally sequentially, co-localizing one or more biologicalassemblers and two or more charged tRNA, and optionally further operableto control the timing and/or order for, optionally sequentially,separating the one or more biological assemblers and the two or morecharged tRNA and/or one or more tRNA.

Unit 4222 is optionally one or more controller units, operable tocontrol the timing for providing and/or removing one or more biologicalassembler components to and/or from one or more identifiable locations.In some embodiments, one or more apparatus includes one or morecontroller units to control an order and/or timing in which each sourceprovides one or more biological assembler components to one or morefourth locations. In some embodiments, one or more apparatus include,but are not limited to, one or more peptide synthesizer units and one ormore controller units that are operable to control the timing forproviding and optionally for removing one or more biological assemblercomponents. In some embodiments, one or more of the one or more peptidesynthesizer units and one or more of the one or more biologicalassembler components controller units are the same unit.

In some embodiments, one or more controller units are operable tocontrol the timing and/or order for providing, optionally sequentially,one or more biological assembler components, and optionally furtheroperable to control the timing and/or order for removing one or morebiological assembler components. In some embodiments, one or morecontroller units are operable to control the timing and/or order for,optionally sequentially, co-localizing one or more biological assemblercomponents with two or more charged tRNA, wherein at least two of thetwo or more charged tRNA are at one or more different locations, andoptionally further operable to control the timing and/or order for,optionally sequentially, separating the one or more biological assemblercomponents from the two or more charged tRNA and/or one or more tRNA. Insome embodiments, one or more controller units are operable to controlthe timing and/or order for, optionally sequentially, co-localizing oneor more biological assembler components and two or more charged tRNA,and optionally further operable to control the timing and/or order for,optionally sequentially, separating the one or more biological assemblercomponents and the two or more charged tRNA and/or one or more tRNA.

Unit 4223 is optionally one or more controller units, operable tocontrol the timing and/or the order for providing and/or removing one ormore nucleic acids to and/or from one or more identifiable locations. Insome embodiments, one or more apparatus include, but are not limited to,one or more peptide synthesizer units and one or more controller unitsthat are operable to control the timing for providing and optionally forremoving one or more nucleic acids. In some embodiments, one or more ofthe one or more peptide synthesizer units and one or more of the one ormore biological assembler components controller units are the same unit.

In some embodiments, one or more controller units are operable tocontrol the timing and/or the order for co-localizing, optionallysequentially, one or more nucleic acids, and optionally further operableto control the timing and/or the order for removing, optionallysequentially, one or more nucleic acids. In some embodiments, one ormore apparatus includes one or more controller units operable to controlan order and/or timing in which each source optionally provides,optionally sequentially, one or more nucleic acids to one or more thirdlocations, and/or optionally removes, optionally sequentially, one ormore nucleic acids from one or more third locations. In someembodiments, one or more controller units are operable to control thetiming and/or the order for providing and optionally for removing one ormore DNA, one or more cDNA, one or more RNA, and/or one or more mRNA. Insome embodiments, the one or more nucleic acids are provided to one ormore identifiable locations and/or to one or more biological assemblers,or one or more biological assembler components.

Unit 4224 is optionally one or more controller units, operable tocontrol the timing for providing and/or removing one or more tRNA, oneor more amino acids, and/or one or more tRNA charging components toand/or from one or more identifiable locations. In some embodiments, oneor more apparatus include, but are not limited to, one or more peptidesynthesizer units and one or more controller units that are operable tocontrol the timing for providing and optionally for removing one or moretRNA, one or more amino acids, and/or one or more tRNA chargingcomponents. In some embodiments, the one or more peptide synthesizerunits and the one or more tRNA charging in controller units are the sameunit. In some embodiments, one or more controller units are operable tocontrol timing and/or order for charging one or more tRNA. In someembodiments, one or more controller units 422 include one or more firstcontroller units 4224 operable to control one or more of an order ortiming in which each source provides one or more tRNA to one or moresecond locations; one or more second controller units operable tocontrol one or more of the order or the timing in which each sourceprovides one or more amino acids to the one or more second locations;and one or more third controller units operable to control one or moreof the order or the timing in which each source provides one or moretRNA charging components to the one or more second locations; andwherein one or more of the one or more first controller units areoptionally the same as one or more of the one or more second controllerunits, and optionally the same as one or more of the one or more thirdcontroller units.

Unit 4225 is optionally one or more controller units, operable tocontrol the timing and/or the order for providing and/or removing one ormore nucleotides and/or one or more nucleic acid synthesis components toand/or from one or more identifiable locations. In some embodiments, oneor more apparatus include, but are not limited to, one or more peptidesynthesizer units and one or more controller units that are operable tocontrol the timing and or the order for providing and optionally forremoving one or more nucleotides and/or one or more nucleic acidsynthesis components. In some embodiments, the one or more peptidesynthesizer units and the one or more nucleic acid synthesis controllerunits are the same unit. In some embodiments, one or more controllerunits are operable to control the timing and/or the order forsynthesizing one or more DNA, one or more cDNA, one or more RNA, and/orone or more mRNA. In some embodiments, one or more apparatus include oneor more first controller units to control one or more of an order ortiming in which each source provides one or more nucleotides to the oneor more third locations; and one or more second controller units tocontrol one more of the order or the timing in which each sourceprovides one or more nucleic acid synthesis components to the one ormore third locations; wherein the one or more first controller units areoptionally the same as the one or more second controller units.

FIG. 6 shows a schematic 400 of illustrative embodiments of theapparatus 410 of FIG. 1, with specific illustrative embodiments of oneor more computing units 426, including unit 4260, unit 4261, unit 4262,unit 4263, unit 4264, and unit 4265, wherein one or more of these unitsare optionally the same unit. In some embodiments, one or more apparatusinclude, but are not limited to, one or more peptide synthesizer units420 and one or more computing units 426. In some embodiments, the one ormore peptide synthesizer units 420 and the one or more computing units426 are the same one or more units. In some embodiments, one or moreapparatus optionally further includes, but is not limited to, one ormore controller units 422, one or more sourcing units 432, and one ormore monitoring units 440. In some embodiments, the one or more peptidesynthesizer units 420, the one or more sourcing units 432, the one ormore controller units 422, the one or more computing units 426, and theone or more monitoring units 440 are the same unit. In some embodimentsone or more of the one or more controller units are optionally the sameas one or more of the one or more computing units.

In some embodiments, one or more computing units 426 are operable todetermine the timing and/or the order for providing and/or co-localizingone or more peptide synthesis components at one or more identifiablelocations, and optionally further operable to determine the timingand/or the order for removing and/or separating one or more peptidesynthesis components from one or more identifiable locations. The one ormore peptide synthesis components include, but are not limited to, oneor more charged tRNA, one or more tRNA, one or more biologicalassemblers, one or more biological assembler components, one or morenucleic acids, and/or nucleic acid synthesizing components.

In some embodiments, the timing is at least partially based on themechanism of peptide synthesis performed by the apparatus. Mechanisms ofpeptide synthesis include, but are not limited to, charged tRNA beingprovided sequentially to one or more locations, charged tRNA beingprovided sequentially to affixed biological assemblers; biologicalassemblers being provided sequentially to affixed charged tRNA; and/orbiological assemblers and charged tRNA being co-localized at one or morelocations. Depending on the mechanism of peptide synthesis, the timingrelated to providing/removing charged tRNA and/or the timing related toproviding/removing biological assemblers, for example, optionallychanges. Timing for each element may be different, and may changedepending on the mechanism of synthesis.

In some embodiments, the timing includes, but is not limited to,sequential timing, fixed timing, variable timing, predicted timing, anddata-driven timing. In some embodiments, the timing is one or moreidentifiable time intervals. In some embodiments, the one or moreidentifiable time intervals are from approximately 0.001 seconds to 0.1seconds and/or approximately 0.01 seconds, or other appropriate timeintervals described elsewhere.

In some embodiments, the order and/or the timing for providing,co-localizing, removing and/or separating one or more peptide synthesiscomponents is at least partially based on a target peptide sequenceand/or a nucleic acid protein coding sequence. In some embodiments, theorder and/or the timing for providing, co-localizing, removing and/orseparating one or more peptide synthesis components is at leastpartially based on a predicted rate of incorporation of two or moreamino acids into one or more peptides, a predicted rate of activity ofone or more biological assemblers, a predicted rate of translocation ofone or more nucleic acids, and/or a predicted rate of release of tRNA.

In some embodiments, one or more computing units 426 are operable todetermine the order and/or the timing for providing, co-localizing,removing and/or separating one or more peptide synthesis components atleast partially based on monitoring of amino acid incorporation into oneor more peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. In some embodiments, one or moremonitoring units 440 are operable to perform the monitoring of aminoacid incorporation into one or more peptides, biological assembleractivity, nucleic acid translocation, and/or tRNA release. In someembodiments, one or more of the one or more monitoring units 440 and oneor more of the one or more computing units 426 are the same units.

In some embodiments, one or more computing units 426 are operable todetermine the order and/or the timing for providing, co-localizing,removing and/or separating one or more peptide synthesis components atleast partially based on measurements including, but not limited to,availability of one or more nucleic acid codons, concentrations of oneor more of the two or more charged tRNA or the one or more tRNA,presence or absence of one or more of the two or more charged tRNA orthe one or more tRNA, or presence or absence of one or more anti-codonson one or more of the two or more charged tRNA or the one or more tRNA.In some embodiments, one or more of these measurements is at leastpartially determined extrinsically. In some embodiments, one or more ofthese measurements are determined based at least partially onmeasurements by one or more monitoring units 440. In some embodiments,one or more of these measurements are provided in real time.

Unit 4260 is optionally one or more computing units, operable fordetermining the timing for providing and/or removing one or more or twoor more charged tRNA and/or released tRNA to and/or from one or moreidentifiable locations. In some embodiments, one or more apparatusinclude, but are not limited to, one or more peptide synthesizer unitsand one or more computing units that are operable to determine thetiming and/or order for providing and optionally for removing one ormore charged tRNA and/or one or more tRNA. In some embodiments, one ormore of the one or more peptide synthesizer units and one or more of theone or more charged tRNA computing units are the same unit.

In some embodiments, one or more computing units are operable todetermine the timing and/or the order for providing, optionallysequentially, two or more charged tRNA to one or more first identifiablelocations, and optionally further operable to determine the timingand/or the order for removing, optionally sequentially, two or morecharged tRNA and/or one or more tRNA from one or more first identifiablelocations. In some embodiments, one or more computing units are operableto determine the timing and/or order for sequentially co-localizing twoor more charged tRNA with one or more biological assemblers, andoptionally sequentially removing two or more charged tRNA and/or one ormore tRNA from the one or more biological assemblers. In someembodiments, one or more computing units are operable to determine thetiming and/or order for sequentially co-localizing two or more chargedtRNA and one or more biological assemblers, and optionally sequentiallyseparating the two or more charged tRNA and/or one or more tRNA and theone or more biological assemblers.

Unit 4261 is optionally one or more computing units operable fordetermining the timing and/or order for providing and/or removing one ormore biological assemblers to and/or from one or more identifiablelocations. In some embodiments, one or more apparatus include, but arenot limited to, one or more peptide synthesizer units and one or morecomputing units that are operable to determine the timing for providingand optionally for removing one or more biological assemblers. In someembodiments, one or more of the one or more peptide synthesizer unitsand one or more of the one or more biological assembler computing unitsare the same unit.

In some embodiments, one or more computing units operable fordetermining the timing and/or order for, optionally sequentially,co-localizing one or more biological assemblers, and optionally furtheroperable to determine the timing and/or order for, optionallysequentially, removing the one or more biological assemblers followingpeptide synthesis. In some embodiments, one or more computing unitsoperable for determining the timing and/or order for, optionallysequentially, co-localizing one or more biological assemblers with twoor more charged tRNA, wherein at least two of the two or more chargedtRNA are at one or more different locations, and optionally furtheroperable to determine the timing and/or order for, optionallysequentially, separating the one or more biological assemblers from thetwo or more charged tRNA and/or one or more tRNA. In some embodiments,one or more computing units operable for determining the timing and/ororder for, optionally sequentially, co-localizing one or more biologicalassemblers and two or more charged tRNA, and optionally further operableto determine the timing and/or order for, optionally sequentially,separating the one or more biological assemblers and the two or morecharged tRNA and/or one or more tRNA.

Unit 4262 is optionally one or more computing units operable fordetermining the timing and/or order for providing and/or removing one ormore biological assembler components to and/or from one or moreidentifiable locations. In some embodiments, one or more apparatusincludes one or more computing units to determine an order and/or timingin which each source provides one or more biological assemblercomponents to one or more fourth locations. In some embodiments, one ormore apparatus include, but are not limited to, one or more peptidesynthesizer units and one or more computing units that are operable todetermine timing and/or order for providing one or more biologicalassembler components to one or more identifiable locations. In someembodiments, one or more of the one or more peptide synthesizer unitsand one or more of the one or more biological assembler componentscomputing units are the same unit.

In some embodiments, one or more computing units operable fordetermining the timing and/or order for providing, optionallysequentially, one or more biological assembler components, andoptionally further operable to determine the timing and/or order forremoving one or more biological assembler components. In someembodiments, one or more computing units operable for determining thetiming and/or order for, optionally sequentially, co-localizing one ormore biological assembler components with two or more charged tRNA,wherein at least two of the two or more charged tRNA are at one or moredifferent locations, and optionally further operable to determine thetiming and/or order for, optionally sequentially, separating the one ormore biological assembler components from the two or more charged tRNAand/or one or more tRNA. In some embodiments, one or more computingunits operable for determining the timing and/or order for, optionallysequentially, co-localizing one or more biological assembler componentsand two or more charged tRNA, and optionally further operable todetermine the timing and/or order for, optionally sequentially,separating the one or more biological assembler components and the twoor more charged tRNA and/or one or more tRNA.

Unit 4263 is optionally one or more computing units, operable fordetermining the timing and/or order for providing and/or removing one ormore nucleic acids to and/or from one or more identifiable locations. Insome embodiments, one or more apparatus include, but are not limited to,one or more peptide synthesizer units and one or more computing unitsthat are operable to determine timing and/or order for providing one ormore nucleic acids to one or more first identifiable locations. In someembodiments, one or more of the one or more peptide synthesizer unitsand one or more of the one or more nucleic acid computing units are thesame unit.

In some embodiments, one or more computing units are operable todetermine the timing and/or the order for co-localizing, optionallysequentially, one or more nucleic acids, and optionally further operableto determine the timing and/or the order for removing, optionallysequentially, one or more nucleic acids. In some embodiments, one ormore apparatus further includes one or more computing units operable todetermine the order and/or the timing in which each source optionallyprovides one or more nucleic acids to one or more third locations, andoptionally removes one or more nucleic acids from one or more thirdlocations. In some embodiments, one or more computing units are operableto determine the timing and/or the order for providing and optionallyfor removing one or more DNA, one or more cDNA, one or more RNA, and/orone or more mRNA. In some embodiments, the one or more nucleic acids areprovided to one or more identifiable locations and/or to one or morebiological assemblers, or one or more biological assembler components.

Unit 4264 is optionally one or more computing units operable fordetermining the timing and/or order for providing and/or removing one ormore tRNA, one or more amino acids, and/or one or more tRNA chargingcomponents to and/or from one or more identifiable locations. In someembodiments, one or more apparatus include, but are not limited to, oneor more peptide synthesizer units and one or more computing units thatare operable to determine timing and/or order for providing one or moretRNA, one or more amino acids, and/or one or more tRNA chargingcomponents to one or more first identifiable locations. In someembodiments, the one or more peptide synthesizer units and the one ormore tRNA charging controller units are the same unit. In someembodiments, one or more computing units are operable to determinetiming and/or order for charging one or more tRNA. In some embodiments,one or more apparatus include one or more first computing units todetermine one or more of an order or timing in which each sourceprovides one or more tRNA to the one or more second locations; one ormore second computing units to determine one or more of the order or thetiming in which each source provides one or more amino acids to the oneor more second locations; and one or more third computing units todetermine one or more of the order or the timing in which each sourceprovides the one or more tRNA charging components to the one or moresecond locations; and wherein one or more of the one or more firstcomputing units are optionally the same as the one or more secondcomputing units, and optionally the same as the one or more thirdcomputing units.

Unit 4265 is optionally one or more computing units, operable fordetermining the timing and/or order for providing and/or removing one ormore nucleotides and/or one or more nucleic acid synthesis components toand/or from one or more identifiable locations. In some embodiments, oneor more apparatus 410 include, but are not limited to, one or morepeptide synthesizer units 420 and one or more computing units that areoperable to determine timing and/or order for providing one or morenucleotides and/or one or more nucleic acid synthesis components 4265 toone or more identifiable locations. In some embodiments, the one or morepeptide synthesizer units 420 and the one or more nucleic acid synthesiscomputing units 4265 are the same unit. In some embodiments, one or morecomputing units 4265 are operable to determine timing and/or order forsynthesizing one or more DNA, one or more cDNA, one or more RNA, and/orone or more mRNA. In some embodiments, one or more apparatus include oneor more first computing units to determine one or more of an order ortiming in which each source provides one or more nucleotides to the oneor more third locations; and one or more second computing units todetermine one more of the order or the timing in which each sourceprovides one or more nucleic acid synthesis components to the one ormore third locations; wherein the one or more first computing units areoptionally the same as the one or more second computing units.

In some embodiments, one or more apparatus 410 include, but are notlimited to, one or more tRNA charging units 428 operable to charge oneor more tRNA with one or more amino acids. In some embodiments, one ormore apparatus 410 include, but are not limited to, one or more peptidesynthesizer units 420 and one or more tRNA charging units 428. In someembodiments, one or more of the one or more tRNA charging units 428 andone or more of the one or more peptide synthesizer units 420 are thesame unit. In some embodiments, one or more tRNA charging units 428 areoperable to co-localize one or more tRNA, one or more amino acids, andone or more tRNA charging components in one or more third identifiablelocations. In some embodiments, the one or more tRNA charging units 428are operable to provide one or more charged tRNA to one or more peptidesynthesizer units and/or to one or more sources of charged tRNA 4320. Insome embodiments, one or more of the one or more third identifiablelocations are the same as one or more of the one or more first locationsand/or the one or more second locations. In some embodiments, one ormore of the one or more third identifiable locations are the same as oneor more sources of charged tRNA 4320. In some embodiments, one or morethird identifiable locations are located in one or more peptidesynthesizer units 420. In some embodiments, the one or more tRNAcharging units 428 include one or more fluid flows.

In some embodiments, one or more apparatus include, but are not limitedin, one or more peptide synthesizer units 420 and one or more tRNAcharging units 428 that are operable to charge one or more natural tRNAwith one or more amino acids, one or more unnatural tRNA with one ormore amino acids, one or more arbitrary tRNA with one or more aminoacids, one or more tRNA with one or more natural amino acids, one ormore tRNA with one or more unnatural amino acids, and/or one or moretRNA with one or more arbitrary amino acids. In some embodiments, theone or more apparatus 410 include, but are not limited to one or moresources of tRNA 4324, one or more sources of amino acids 4326, and/orone or more sources of tRNA charging components 4325.

In some embodiments, one or more apparatus include one or more nucleicacid synthesizer units 430 operable to synthesize nucleic acids. In someembodiments, one or more apparatus 410 include, but are not limited to,one or more peptide synthesizer units 420 and one or more nucleic acidsynthesizer units 430. In some embodiments, one or more of the one ormore peptide synthesizer units 420 and one or more of the one or morenucleic acid synthesizer units 430 are the same unit. In someembodiments, one or more nucleic acid synthesizer units 430 are operableto synthesize one or more RNA, one or more mRNA, one or more cDNA,and/or one or more DNA. In some embodiments, one or more nucleic acidsynthesizer units 430 are operable to co-localize one or morenucleotides and/or one or more nucleic acid synthesis components in oneor more fourth identifiable locations. In some embodiments, one or morenucleic acid synthesis components include, but are not limited to, oneor more DNA synthesis components, one or more cDNA synthesis components,one or more RNA synthesis components, and one or more mRNA synthesiscomponents.

In some embodiments, one or more of the apparatus 410 include, but arenot limited to, one or more nucleic acid synthesizer units 430 operableto provide one or more nucleic acids to one or more peptide synthesizerunits 420. In some embodiments, one or more of the one or more fourthidentifiable locations are optionally the same as one or more of the oneor more first identifiable locations, and/or one or more sources ofnucleic acids 4321. In some embodiments one or more fourth identifiablelocations are located in one or more peptide synthesizer units 420. Insome embodiments, the one or more nucleic acid synthesizer units 430include one or more fluid flows.

EXAMPLES

The following Examples are provided to illustrate, not to limit, aspectsof the present invention. Materials and reagents described in theExamples are commercially available unless otherwise specified.

Example 1 Arbitrary Synthesis of Polypeptides—Basic Protocol

Ribosomal assemblies, either derived from a cell lysate or reconstitutedfrom one or more individual components, are incubated with a nucleicacid sequence, for example mRNA, either before or after addition to areaction chamber to form an initiation complex. Then, assembly of atarget polypeptide is achieved through sequentially combining theribosome initiation complex with appropriate aminoacylated tRNAs(aa-tRNA; charged tRNA) using one of several methods described herein orbelow.

For example, aminoacylated tRNAs may be added sequentially to thereaction chamber such that the amino acids are provided in the order ofthe target protein sequence and the anti-codons align with the codons ofthe template nucleic acid. After a fixed or variable interval, unusedaa-tRNA and used deacylated tRNA may be cleared and the next aa-tRNA inthe sequence added to the ribosomes. Sequential additions of aa-tRNAsare continued until termination of translation has been achieved. Thetranslated polypeptide is optionally isolated from the translation mix.

Alternatively, the ribosome complex may be exposed sequentially toaa-tRNA such that the amino acids are available in the order of thetarget protein sequence and the anti-codons align with the codons of thetemplate nucleic acid. After a fixed or variable interval, the ribosomecomplex with nascent polypeptide strand may be separated from unusedaa-tRNA and used deacylated tRNA, and then exposed to the next aa-tRNAin the sequence. Sequential exposure of the ribosome complex includingthe nascent polypeptide strand to aa-tRNAs is continued untiltermination of translation has been achieved. The translated polypeptidemay be isolated from the translation mix.

Translation is terminated once the nucleic acid template has reached theend of the target coding sequence. In a cell free lysate, terminationand release of the nascent polypeptide chain may occur once the ribosomeassembly has reached a series of one or more stop codons and noadditional aa-tRNAs are added to the system. Under these conditions,release factors associated with the cell free lysate facilitatetermination and release of the polypeptide. In a reconstituted system,termination and release of the nascent polypeptide chain is facilitatedby the addition of release factors to the reaction once the ribosomeassembly has reached a series of one or more stop codons and noadditional aa-tRNAs are added to the system.

Example 2 Timing of Sequential Addition of Each aa-tRNA

The addition and incubation of each aa-tRNA in the translation reactioncan be set to a uniform interval or a variable interval. The uniforminterval can be set, for example, from 0.001 to 0.1 seconds for eachaa-tRNA. The rate of translation with natural amino acids in anoptimized in vitro system is approximately 10 codons per second (Arch.Biochem. Biophys. (1996) 328:9-16).

A variable interval which might, for example, be dependent upon thespecific aa-tRNA can be determined empirically for each individualaa-tRNA or can be determined in real time during translation.Fluorescence resonance energy transfer (FRET) can be used to measuretRNA interactions within the ribosomes. This type of analysis can beused to determine the kinetics of ribosome interaction for each of thesynthesized aa-tRNAs, and these numbers used, for example, to set thetime interval for addition and clearance of each individual aa-tRNA.

For example, FRET can be used to measure the interaction of tRNA speciesin the P and A sites of the ribosome as described by Blanchard et al.(Nat. Struct. Mol. Biol. (2004) 11:1008-1014). E. coli ribosomes areinitiated in vitro with fluorescently labeled fMet-tRNA^(fMet) (Cy3) inthe P site and tethered to a streptavidin derivatized quartz microscopeslide via biotinylated mRNA. The aa-tRNA of interest, for examplePhe-tRNA^(Phe), is labeled with Cy5 on a naturally occurring modifiednucleotide (acp³U/position 47) using standard procedures. Phe-tRNA^(Phe)(Cy5) is complexed with the elongation factor ET-Tu as previouslydescribed (J. Biol. Chem. (2005) 280:36065-36072) and immediately addedby stopped-flow delivery to the surface-immobilized ribosomes.

A total internal reflection (TIR) fluorescence microscope may be used tomeasure the changes in Cy3 and Cy5 fluorescence as the labeled tRNAsmove into proximity of one another within the ribosome. Maximal FRETsignal will occur when, for example, Phe-tRNA^(Phe) (Cy5) is in the Asite, has been released from ET-Tu, and is in close proximity tofMet-tRNA^(fMet) (Cy3) during peptide bond formation (Nat. Struct. Mol.Biol. (2004) 11:1008-1014). The time to reach this maximal signal can beused to determine, for example, the time interval required for aspecific aa-tRNA to bind to the ribosome, transfer the associated aminoacid, and dissociate from the ribosome. Similar FRET analysis can bedone, for example, using labeled ribosomes and aa-tRNA (Nucleic AcidsRes. (2005) 33:182-189).

Alternatively, empirical rates of amino acid incorporation for eachaa-tRNA can be determined for example by using a puromycin assay systemas described by Beringer et al. (J. Biol. Chem. (2005) 280:36065-36072).This assay measures the rate of peptidyl transferase (i.e. transfer ofamino acid to a nascent peptide chain) by monitoring transfer of thegrowing peptide chain to puromycin.

As an example, fMet-aa-tRNA^(Met) labeled with Cy3 on the Met is loadedinto the P site of ribosomes and this complex is immobilized on asurface as described by Blanchard et al. (Proc. Nat. Acad. Sci. (2004)101:12893-12898). Association of the Cy3 label with the surfacerestricted ribosomes creates surface localized fluorescence. Uponaddition of puromycin, the Cy3-Met-aa-tRNA^(Met) is transferred to thepuromycin which is quickly released from the ribosome and thesurface-localized fluorescence disappears. The rate at whichfluorescence disappears can be used to determine the rate at which apeptide bond forms for a given amino acid.

The calculated empirical rate at which a given tRNA either binds to oris released from the ribosome assembly can be used to modulate the timeinterval over which specific aa-tRNAs are sequentially added to fixedribosomes or over which ribosomes are moved sequentially passed fixedaa-tRNAs. These calculated rates can also be used to modulate the timeinterval over which free aa-tRNAs and ribosomes interact.

Real-time Assessment of Translation

Real-time assessment of translation progression with a specific aa-tRNAcan be measured and a feedback loop used to indicate when the nextaa-tRNA should be added. Various components of the translation systemcan be monitored for change during each cycle.

For example, the release of phosphate (Pi) from the hydrolysis of GTP toGDP during translation can be used to monitor progression of theprocess. GTP is hydrolyzed to GDP by a conformation change in theelongation factor/aa-tRNA ternary complex once the complex has bound tothe ribosome. In addition, GTP is hydrolyzed to GDP by an elongationfactor during the process of translocation and removal of deacylatedtRNA from the ribosomal complex.

Release of Pi can be measured using a phosphate binding protein assay.Phosphate binding protein (PBP) from E. coli, for example, is purifiedas described (Methods (2005) 37:183-189). Alternatively, PBP can becloned from, for example, the E. coli phoS gene by standard molecularbiology techniques. Alternatively, cDNA encoding PBP from, for example,E. coli can be synthesized de novo by a commercial source (e.g. BlueHeron Biotechnology, Bothell, Wash.), based on the published nucleotidesequence (J. Bacteriol. (1984) 157:909-917).

Purified PBP is labeled, for example, with a fluorescent dye such as7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)))) couramin(MDCC) as described previously (Methods (2005) 37:183-189). Briefly, 100μM PBP is incubated with 150 μM MDCC, 5 μM MnCl2, 200 μM7-methylguanosine, 1 μM glucose 1,6-bisphosphate, and 0.2 U/ml purinenucleoside phosphorylase in 20 mM Tris-HCl (pH 8.2) in the dark for 45min at room temperature. The MDCC-PBP solution is then passed over aBioGel size exclusion column to separate the labeled protein from thesmaller reaction components. PBP is extremely stable and once conjugatedto MDCC can be stored at −80° C. for years (Methods (2005) 37:183-189).Alternatively, PBP can be labeled with other fluorescent dyes such asrhodamine (Biochemistry (2006) 45:14764-14771).

Alternatively, a PhosphoSensor system for measuring Pi via aphosphate-binding protein is commercially available (e.g. Invitrogen,Carlsbad, Calif.). The binding of Pi to MDCC-labeled PBP increases thefluorescence emission by as much as 12 fold at a wavelength of 464 nmwhen the complex is excited at 425 nm (Methods (2005) 37:183-189). FreshGTP and labeled PBP are added coincident with each tRNA addition. Astranslation proceeds during a given cycle, the Pi associatedfluorescence increases to a plateau level. At the end of a given cycle,under conditions where the ribosomes are fixed or size excluded, thereaction chamber is flushed of unused aa-tRNA and deacylated tRNA andthe next aa-tRNA is added to the chamber with additional GTP and labeledPBP. Alternatively, under conditions where the aa-tRNAs are fixed ortethered, the ribosomes move away from the unused aa-tRNA and deacylatedtRNA to a new location in the reaction chamber to interact with the nextaa-tRNA in the presence of additional GTP and labeled PBP.

Alternatively, real-time assessment of translation can be monitored bymeasuring depletion of aa-tRNA or appearance of deacylated tRNA. Forexample, once tRNA is deacylated, the acceptor stalk at the 3′end of thetRNA sequence is revealed and becomes accessible to hybridization by,for example, a peptide nucleic acid (PNA) labeled, for example, withbiotin, digoxigenin, fluorescent dyes, or reporter enzymes. Sequencespecific Cy3-labeled PNAs are available through custom synthesisservices (e.g. Panagene, Daejeon, Korea).

The selective PNA can be bound to a biosensor that emits a signal oncebinding with the deacylated tRNA occurs. For example, evanescent fieldfluorescence can be used to measure fluorescence changes at the surfacein response to Cy3-deacylated tRNA binding to immobilized PNA.Alternatively, the tRNA and the PNA can be labeled with Cy3 and Cy5,respectively, and FRET used to measure the interaction of the twospecies. Molecules can be labeled with Cy3 and Cy5 using commerciallyavailable labeling kits (e.g. Amersham Biosciences, Piscataway, N.J.).

Interaction of PNA and deacylated tRNA can be measured without labelingthe components by, for example, surface plasmon resonance. The specificPNA is immobilized on a surface plasmon resonance-based biosensor andthe change in mass associated with binding of deacylated tRNAs aremonitored.

A selective PNA can be synthesized using an Applied Biosystems 3400 DNASynthesizer or an ABI 3900 Synthesizer, or using custom commercialservices (e.g. Panagene, Daejeon, Korea). The PNA is further purified byreverse phase HPLC on an Ultrasphere ODS C-18, 4.6*250 mm column(Beckman) using an acetonitrile/TFA gradient (from A to B linear in 30min: A, water/0.1% TFA; B, acetonitrile/0.1% TFA) as described byJankowsky et al. (Nucleic Acids Res. (1997) 25:2690-2693).

Under conditions in which the aa-tRNA is fixed or tethered to a surface,the transition from the aa-tRNA to the deacylated state can be measuredbased on the change in mass of the deacylated tRNA using a MEMS device(see, e.g. U.S. Pat. No. 6,722,200 and U.S. Pat. No. 6,457,361).Similarly, if the ribosome complex is tethered, the docking of aa-tRNAand release of the amino acid and the deacylated tRNA can be measuredbased on the change in mass.

Example 3 Ribosomal Complexes for the Arbitrary Synthesis ofPolypeptides

The ribosome system used in the translation reaction may be derived fromcell free lysates or from reconstitution of purified native orrecombinant components. Cell-free protein synthesis systems consistingof a cell extract are prepared, for example, from E. coli, wheat germ orrabbit reticulocytes.

The cell lysate is prepared, for example, from rabbit reticulocytesfollowing the protocol of Jagus et al. (Current Protocols in CellBiology (1998) Juan S. Bonifacino, Mary Dasso, Joe B. Harford, JenniferLippincott-Schwartz, and Kenneth M. Yamada (eds.) John Wiley & Sons,Inc). Whole blood is centrifuged for 10 minutes at 1400×g andsubsequently lysed for 5 minutes with RNase-free distilled water. Theresulting lysate is centrifuged for 20 minutes at 20,000×g to removecell membranes and mitochondria. The resulting supernatant contains allof the protein components necessary to accomplish protein synthesis inthe presence of an added nucleic acid template. Cell-lysates for invitro translation are also available from commercial sources (e.g.Invitrogen, Carlsbad, Calif.; Ambion, Austin, Tex.).

For some syntheses, the lysate is depleted of endogenous aa-tRNA.Depletion of aa-tRNA from a commercially available rabbit reticulocytelysate (Invitrogen, Carlsbad, Calif.) is accomplished by passing thelysate over a 1 ml ethanolamine-sepharose column equilibrated with 25 mMKCl, 10 mM NaCl, 1.1 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 10 mMHEPES—KOH (pH 7.4) supplemented with 80 mM potassium acetate and 0.5 mMmagnesium acetate to match the ionic concentration of the lysate. Underthese conditions, aa-tRNA has been shown to selectively bind to thecolumn matrix (RNA (2001) 7:765-773).

Alternatively, a reconstituted ribosomal complex can be used thatcontains ribosomes, various protein factors, and the nucleic acidtemplate of interest. An example of a reconstituted ribosomal complexincludes hexahistadine-tagged recombinant initiation factors (IF1, IF2,and IF3), elongation factors (EF-G, EF-Tu, and EF-Ts), release factors(RF1, RF2, and RF3), and termination factor RRF generated by PCR,expressed in E. coli and purified using a nickel-chelating resin thatbinds the His tag (Nature Biotech. (2001) 19:751-755; Methods (2005)36:299-304). NTPs, creatine phosphatase, creatine kinase, myokinase, andnucleoside diphosphate kinase, for example, are added as part of anenergy-recycling system. Ribosomes are isolated from E. coli, forexample, by centrifugation of a cell lysate at 30,000×g followed byrepeated centrifugation of the 30,000×g supernatant at 100,000×g andwashing in 10 mM Tris-acetate, pH 8.2, 14 mM magnesium acetate, 60 mMpotassium acetate, 1 mM dithiothreitol, and 1 M ammonium chloride(Methods (2005) 36:279-290).

The ribosome complex may be free in translation solution, but retainedin a “flow cell” reaction chamber by size exclusion mediated by, forexample, the limiting size of an out flow channel. Alternatively, theribosome complex may be selectively retained by a semi-permeablemembrane. Semi-permeable membranes include those available fromcommercial sources (e.g. Millipore, Bellerica, Mass.) for example Biomaxor Amicon PM high flow ultrafiltration membranes. Membranes are chosenwith molecular weight cut-offs that would retain the ribosome complexbut allow free-flow of added tRNA, for example 30-50 kilodalton (kDa).

Alternatively, ribosomes may be attached to a solid surface. In oneexample, the solid surface may be a thin mica sheet that forms, forexample, one or more surfaces of a flow cell. 70S ribosomes may beattached non-specifically to the mica sheet. In one method, ribosomesare incubated for 5 minutes with the mica sheet in a buffer having, forexample, 5 mM KH₂PO₄, pH 7.3, 95 mM KCl, 5 mM NH₄Cl, 5 mM Mg acetate,0.5 mM CaCl₂, 8 mM putrescine, 1 mM spermidine, 1 mM dithiothreitol. Theattached ribosomes are washed 3 times with 50 mM Tris-HCl, pH 7.4, 50 mMKCl, 10 mM Mg Cl₂, washed 1 time with protein-based blocking solutionsuch as 5% bovine serum albumin treated with an RNase inhibitor,followed by 3 final washes with buffer. To the ribosomes are then addedthe template nucleotide and for example purified elongation factors aswell as components of an energy recycling system. (RNA (2003)9:1174-1179).

Alternatively, ribosomes may be attached to a glass surface (e.g., amicroscope cover slip) by a chemical reaction (Biophys. J. (2005)89:1909-1919). Glass cover slips may be functionalized with amine groupsusing, for example, 3-aminopropyltriethoxysilane (APTES, United ChemicalTechnologies, Bristol, Pa.). The cover slips are then treated withN-[k-maleimidoundecanoyloxyl]sulfosuccinimide ester (Sulfo-KMUS);Pierce, Rockford, Ill.), a heterobifunctional cross-linker which carriesa succinimide group at one end and a maleimide group at the other end,separated by a 11 carbon linker chain. The succinimide group reacts withthe amines on the glass surface, leaving the maleimide groups exposedand reactive toward protein sulfhydryls at the surface of the ribosome.The length of the linker carbon chain can be varied by usingcommercially available sulfo-NHS reagents with spacer arms ranging forexample from 4-16 angstrom (e.g. Pierce, Rockford, Ill.).

Ribosomes may also be immobilized via association with an affixed strandof nucleic acid sequence, for example 5′ or 3′ biotinylated mRNA. ThemRNA is biotinylated at the 5′ end, for example, by a one steptranscription procedure using biotinylated AMP or GMP as a transcriptioninitiator (Nucleic Acids Res. (2005) 33:e129). Alternatively,nucleotides can be biotinylated using, for example, EZ-Link PFP-Biotin((+)-Biotin pentafluorophenyl-ester) per the instructions provided bythe manufacturer (TFP-PEO-Biotin; Pierce, Rockford, Ill.).

The 70S ribosome complexes are initiated on the biotinylated mRNA invitro in 50 mM Tris acetate (pH 7.5), 100 mM KCl, 5 mM ammonium acetate,0.5 mM calcium acetate, 5 mM magnesium acetate, 6 mM 2-mercaptoethanol,5 mM putrescine, and 1 mM spermidine, and subsequently purified bysucrose density ultracentrifugation in the above buffer with 20 mMmagnesium acetate as described by Pavlov & Ehrenberg (Arch. Biochem.Biophys. (1996) 328:9-16). The resulting initiation complexes containingthe biotinylated mRNA are immobilized, for example, on the surface of aquartz microscope slide coated with a combination of polyethylene glycol(PEG) and streptavidin-biotin-PEG. Alternatively, the biotinylatedribosome complex is bound to the surface of a microchip or a polystyrenebead coated with streptavidin. The surfaces are further treated with ablocking solution containing 10 μM BSA/10 μM double-stranded DNA.

Example 4 Nucleic Acid Templates for the Arbitrary Synthesis ofPolypeptides

The nucleic acid template used in the translation reaction can be DNA,RNA or mRNA, and may be, for example, a traditional protein codingnucleic acid sequence, or an arbitrary nucleic acid sequence used as atemplate for arbitrary protein synthesis. The nucleic acid sequence maycontain a ribosomal binding site, for example, a Shine-Dalgamo sequenceas well as an initiation codon, for example AUG.

Traditional mRNA used in the translation reaction may be transcribedfrom a cDNA construct containing the nucleotide sequence correspondingto the polypeptide sequence of interest. The cDNA construct containspolymerase promoter sequences allowing for runoff transcription using,for example, the T7 RNA polymerase with standard reagents available fromcommercial sources (e.g. Stratagene, La Jolla, Calif.).

The cDNA corresponding to the polypeptide of interest can be derivedfrom screening of cDNA libraries, amplification of cDNA libraries orgenomic DNA using specific oligonucleotide primers and the polymerasechain reaction (PCR), or cDNA purchased intact from commercial sources(e.g. Origene, Rockville, Md.). DNA may also be synthesized de novousing custom commercial services (e.g. Blue Heron Biotechnology,Bothell, Wash.).

As an illustrative example, the arbitrary nucleic acid template iscomposed of a single nucleotide. Synthesis and use of high-molecularweight poly(U) (average length 8,000-10,000 bases) is known in the artand is prepared by polynucleotide phosphorylase polymerization of UDPand fractionated by Sephacryl column chromatography (Biophys. J. (2005)89:1909-1919). In this instance, only one, two or three tRNA species areneeded, with a three-base, four-base, or five-base anticodon containingthree, four, or five As, respectively. A variety of natural andunnatural amino acids (optionally including the entire set of naturalamino acids) are acylated to these tRNA species.

Alternatively, the arbitrary nucleic acid sequence could be composedentirely of alternating or repeating stop codons as described herein.Alternatively, the arbitrary nucleic acid sequence could be composed ofa set of optionally repeating 3-base, 4-base or 5-base codons which arenot stop codons. In general, any subset of the natural codons can beused to create the nucleic acid sequence (2, 3, 4, 5, 6, 7, 8, 9, 10,etc.), so long as the corresponding tRNAs are available and acylatedwith the required natural and/or unnatural amino acids. The codons maybe used in a repeating or a non-repeating pattern. The nucleic acidsequence may also be optimized for codon usage and easy translation, aswell as to avoid using the same codon for different amino acids when thecodons are adjacent, or optionally within one, two, three, four, five,six, seven, eight, nine, or ten codons of the same codon among otherthings.

Example 5 Recovery of Translation Product from the Arbitrary Synthesisof Polypeptides

At the end of translation, the peptide or protein product may berecovered from the reaction vessel. Protein can be isolated in a varietyof ways, including by virtue of a small tag engineered into the proteinsequence.

Hexahistidine, a string of 6 histidines, is inserted, for example, atsome point along the length of the protein sequence. The polyhistidinebinds strongly to divalent metal ions such as nickel and cobalt and canbe immobilized on, for example, a matrix or surface containing nickelions. After extensive washing to remove unbound material, the boundHis-tagged protein is eluted from the matrix or surface with, forexample, imidazole or low pH (Nucleic Acids Res. (1991) 19:6337-6338).

Alternative methods for tagging the protein include, for example, a Mycepitope tag (Mol. Cell. Biol. (1993) 13:5861-5876) or some other smallepitope or recognition sequence recognized by a specific antibody oraptamer.

Alternatively, tags associated with the components of a reconstitutedribosomal complex can be used to selectively separate these componentsfrom the un-tagged translation product. At the completion oftranslation, large particles, for example, a microsphere, are introducedinto the reaction vessel. The particles have associated nickel or cobaltaffixed to their surface which then binds the His-tagged proteins.Particles of this type are available from commercial sources (e.g.Dynabeads® TALON™, Invitrogen, Carlsbad, Calif.). Flow is directedtowards an outlet that excludes both the large ribosome complex and theparticles, allowing the un-tagged protein to be expelled.

Example 6 aa-tRNAs for the Arbitrary Synthesis of Polypeptides

There are 20 common amino acids that are naturally incorporated intopolypeptides during translation in eukaryotes and that can be used inthe methods and apparatus described herein. In addition, incorporationof an unnatural amino acid into a polypeptide sequence is possible, forexample, by addition of the unnatural amino acid on a natural tRNA. Bothunnatural and natural amino acids may also be incorporated into apolypeptide sequence using unnatural tRNA, for example tRNA withmodified anti-codon sequences. During translation, the natural (ormodified) aa-tRNA then places the unnatural (or natural) amino acid intothe elongating polypeptide sequence based on the anticodon sequence ofthe aa-tRNA and the respective codon sequence within the nucleic acidtemplate.

Each aa-tRNA may be added to the translation reaction by itself, oralready pre-complexed with an elongation factor, for example ET-Tu. Theformation of this complex is initiated, for example, by incubation ofEF-Tu•GDP (˜300 pmol) with GTP (1 mM), phosphoenol pyrovate (3 mM), andpyruvate kinase (100 μg/ml) in 20 μl of 50 mM Tris-HCl (pH 7.5), 40 mMNH4Cl, 10 mM MgCl2, and 1 mM DTT for 10 min at 37° C. to exchange thebound GDP with GTP. Equimolar aa-tRNA is added in the same buffer andfurther incubated for 5 min at 37° C. (J. Biol. Chem. (2005)280:36065-36072).

The aa-tRNA/ET-Tu complex may be free in solution or tethered to asubstrate, for example, via interaction of ET-Tu with an affixedanti-ET-Tu antibody. Upon engagement of aa-tRNA in ribosome, GTP isconverted back to GDP and the aa-tRNA/ET-Tu complex is broken.

The aa-tRNAs may be immobilized using a variety of methods including,for example, by electrostatic binding to poly-L-lysine. Alternatively,biotinylated aa-tRNAs may be immobilized by binding to avidin orstreptavidin on the surface of the reaction chamber or to beads, forexample.

Another option is to immobilize the aa-tRNAs via a linker molecule. Forexample, the aa-tRNAs can be immobilized through hybridization to, forexample, the T-loop of the tRNA, with a selective peptide nucleic acid(PNA) (see, e.g. Methods (2005) 36:270-278; Bioorg. Med. Chem. Lett.(2006) 16:4757:4759). The PNA can be bound through a linker to thesurface of the chamber or beads, among others. A selective PNAconjugated to a peptide can be synthesized, for example, using customservices (e.g. Panagene, Daejeon, Korea). The PNA-peptide is linked tothe surface of the chamber or beads by, for example,N-hydroxysuccinimide (NHS) ester (see, e.g. Trends Biotechnol.22:617-622 (2004)).

Alternatively, the aa-tRNA is modified, for example, with Cy3 or Cy5 asdescribed herein and subsequently attached to the surface of thereaction chamber or beads, for example, via an immobilized anti-Cy3/Cy5monoclonal antibody (Sigma Aldrich).

Alternatively, aa-tRNAs may be immobilized in association with anelongation factor. For example, ET-Tu is biotinylated using standardprocedures and is attached to a streptavidin-modified surface of thereaction chamber or beads, for example. aa-tRNA is subsequently bound toET-Tu in a GTP-dependent manner as described herein. Alternatively,aa-tRNA/ET-Tu complexes are generated external to the reaction chamberand then added to the streptavidin-modified reaction chamber.Alternatively, the aa-tRNA/ET-Tu complexes may be attached, for example,via an anti-ET-Tu polyclonal antibody (e.g., BET-A300-957, Axxora LifeSciences Inc. San Diego, Calif.) immobilized to the reaction chambersurface (or surface of beads, among others) using standard procedures.

Synthesis of Natural tRNAs

tRNA species with anti-codons corresponding to the complement of naturalcodons on mRNA can be generated de novo using standard molecular biologytechniques. tRNA nucleotide sequence information for a variety oforganisms is available in public databases (e.g.http://www.staff.uni-bayreuth.de/).

Phenylalanine (Phe) specific tRNA is generated, for example, byannealing complementary oligomers containing the nucleotide sequence oftRNA(Phe). The complementary oligomers may be synthesized using anApplied Biosystems 3400 DNA Synthesizer or an ABI 3900 Synthesizer, orusing custom commercial services. The oligomers are designed to containrestriction sites at the 5′ and 3′ ends, for example Kpn and HindIII,enabling ligation of the fragment into a transcription compatiblevector, such as Bluescript® II KS +/− (Stratagene, La Jolla, Calif.).The fragment is oriented 5′ to 3′ relative to an RNA polymerase promotersequence within Bluescript® II KS +/−, such as that for T7 polymerase.Additional restrictions sites, for example BstNI and FokI, areengineered into the 3′ end of the sequence within the acceptor stem.Linearization of the construct at BstNI or FokI followed by T7polymerase mediated run-off transcription generates tRNA(Phe) witheither an intact acceptor stem compatible for example with enzymaticaminoacylation or a truncated acceptor stem lacking the 3′ terminal CAcompatible for example with chemical aminoacylation (Nucleic Acids Res.(1990) 18:83-88).

Alternatively, a transcription template for tRNA synthesis can begenerated in the absence of cDNA cloning by generating twooligonucleotides of disparate size (Korencic et al. Nucleic Acids Res.(2002) 30:e105). The first and larger oligonucleotide comprises the tRNAgene. The second is for example 23 nucleotides and complementary to the3′ end of the first oligonucleotide. The two oligonucleotides areannealed, forming a double stranded T7 promoter site. Transcriptionusing, for example, the aforementioned template is carried out in areaction containing 40 mM Tris-HCl, pH 8.0, 22 mM MgCl₂, 1 mMspermidine, 5 mM DTT, 0.5% Triton-X100, 4 mM each NTP, 5 mM GMP and 30nM T7 RNA polymerase for 3 h at 37° C. The reaction mix is extractedwith phenol/chloroform and ethanol precipitated. The tRNA issubsequently purified on and extracted from a 12% polyacrylamide gel.

Methods for purifying tRNA species from native sources such as E. coliand yeast have also been described (J. Biol. Chem. (1968)243:5761-5769). Alternatively, purified native tRNA species areavailable from commercial sources (e.g. Sigma-Aldrich, St. Louis, Mo.).

Synthesis of Unnatural tRNAs

Unnatural tRNAs may be defined as tRNAs with primary nucleotide sequencenot normally found in nature and may be synthesized de novo or derivedfrom natural tRNA by, for example, site-directed mutagenesis.

Unnatural tRNA can be synthesized by site-directed mutagenesis of anatural tRNA. Site-directed mutagenesis can be used to modify, forexample, the anticodon region, amino acid acceptor stem, or other partsof the primary tRNA sequence of, for example, tRNA^(Gln). A cDNAconstruct containing the tRNA^(Gln) sequence is generated using, forexample, one of the methods described herein. Point mutations in theprimary sequence are generated using user-defined oligonucleotides of,for example, 20 bases and a commercially available site-directedmutagenesis kit (e.g. QuikChange® XL, Stratagene, La Jolla, Calif.).

Alternatively, unnatural tRNA can be synthesized de novo using themethods described herein, including the two oligonucleotide primerstrategy above and the overlapping oligonucleotides with primerextension strategy described in Example 8 (Methods (2005) 36:270-278).Alternatively, the nucleotide sequence for an unnatural tRNA can besynthesized de novo using custom commercial services (e.g. Blue HeronBiotechnology, Bothell, Wash.).

Aminoacylation of Natural and Unnatural tRNA with Natural Amino Acids

Charged or acylated tRNAs (aa-tRNA) used in the translation reaction aregenerated by addition of an amino acid to a tRNA via enzymatic orchemical aminoacylation. Enzymatic aminoacylation may be achieved usingthe aminoacyl tRNA synthetases specific for each of the twenty naturalamino acids.

For example, phenylalanyl-tRNA^(Phe) can be generated in vitro byenzymatic aminoacylation following the methods of Sampson & Uhlenbeck(PNAS (1988) 85:1033-1037). tRNA^(Phe) is heated to 60° C. and slowlycooled to 25° C. prior to addition of aminoacylation reaction mixture.To the tRNA is added 30 mM Hepes KOH (pH 7.45), 10 μM phenylalanine, 2mM ATP, 15 mM MgCl₂, 25 mM KCl, and 4 mM dithiothreitol. The reaction isinitiated by addition of phenylalanyl-tRNA synthetase at 0.07 to 0.3units/ml. The reaction is carried out for 4 minutes at 37° C.

Enzymatic aminoacylation using aminoacyl tRNA synthetases can also beused to incorporate natural amino acids into unnatural tRNA. Forexample, unnatural tRNA containing a four-base anticodon can byaminoacylated by endogenous E. coli aminoacyl-tRNA synthetases (NucleicAcids Res. (2006) 34:1653-1662).

Amino acid specific aminoacyl tRNA synthetases can be purified from E.coli and yeast, for example, (e.g. Nucleic Acids Res. (1986)14:7529-7539) or through standard cDNA cloning techniques (e.g. NucleicAcids Res. (1998) 26:521-4). Alternatively, full-length cDNA encodingvarious aminoacyl tRNA synthetases are commercially available (e.g.Origene, Rockville, Md.) or can be synthesized de novo (e.g. Blue HeronBiotechnology, Bothell, Wash.) based on published sequence information.

Chemical aminoacylation of natural and unnatural tRNA may be achievedusing for example aminoacylated pdCpA derivatives (Nucleic Acids Res.(1989) 17:9649-9660), or an N-pentenoyl protecting group (Methods (2005)36:245-251).

For example, phenylalanyl-tRNA^(Phe) can be generated by chemicalaminoacylation using the N-pentenoyl protection method as described byLodder et al. (Methods (2005) 36:245-251). Pentenoic acid (49 mmol) andN-hydroxysuccinimide (49 mmol) in CH₂Cl₂ are added toN,N′-dicyclohexylcarbodiimide (50 mmol). After 1.5 hours at roomtemperature, the reaction product is passed over a silica column, elutedwith 7:3 hexane-ethyl acetate, and crystallized from ether-petroleumether (35-60° C.). The resulting 4-pentenoyloxy succinimide ester isincubated with 1 mmol phenylalanine and 2 mmol NaHCO₃ in 1:1H₂O:dioxane. After 16 hours at room temperature, the reaction mixture isdiluted with 1N NaHSO₄ and extracted with ethyl acetate. The organicextract is dried (MgSO₄) and concentrated under diminished pressure. Thecrude product is redissolved in acetonitrile and chloroacetonitrile andincubated for 20 hours at room temperature. The reaction mixture isdiluted with ethyl acetate, washed with 1 N NaHSO₄, and the organicextract dried (MgSO₄) and concentrated under diminished pressure. Thecrude product is applied to a silica gel column and eluted with 4:1ethyl acetate-hexanes. The resulting N-4-pentenoyl amino acidcyanomethyl ester is added to a solution of the tris(tetrabutylammonium)salt of pdCpA in DMF. The reaction mixture is incubated for 3 hours atroom temperature, diluted in 1:2 CH₃CN-50 mM NH₄OAc (pH 4.5) andpurified on a semi-preparative C₁₈ reversed phase column, resulting inN-4-pentenoyl-aminoacyl pdCpA. The last step in this chemicalaminoacylation procedure involves ligation of the aminoacyl-pdCpA totRNA^(Phe) lacking the 3′-terminal pCpA in 50 mM Hepes buffer (pH 7.5)containing 15 mM MgCl₂, 0.75 mM ATP, 10% DMSO, and 100 units of T4 RNAligase. After incubation for 1 hour at 37° C., theN-(4-penenoyl)-phenylalanyl-tRNA is precipitated with ethanol and theresulting pellet dissolved in H₂O. Removal of the pentenoyl protectiongroup prior to translation is accomplished by incubating the tRNA withI₂ (25 mM in 1:1 THF-H₂O). The aminoacyl-tRNA is purified by ethanolprecipitation and can be stored dissolved in buffered aqueous solutionat pH 5.0 at low temperature for several weeks (Methods (2005)36:245-251).

Alternatively, a precursor tRNA with self-aminoacylation activity may beused to aminoacylate itself (EMBO (2001) 20:1797-1806).

Aminoacylation of Natural and Unnatural tRNA Using Unnatural Amino Acids

Unnatural amino acids are known in the art including, but not limitedto, those containing spectroscopic probes, post-translationalmodification, metal chelators, photoaffinity labels, D-enantiomers, aswell as other functional groups and modified structures (e.g. Methods(2005) 36:227-238; Ann. Rev. Biochem. (2004) 73:147-176; Science (2003)301:964-967; Royal Society of Chemistry (2004) 33:422-430). A variety ofunnatural amino acids are available from commercial sources (e.g.Sigma-Aldrich, St. Louis Mo.; EMD Biosciences, San Diego, Calif.).

Naturally occurring aminoacyl tRNA synthetases may be used toincorporate unnatural amino acids into natural tRNAs. For example,homoallylglycine and trifluoroleucine can be incorporated into proteinusing wild type aminoacyl tRNA synthetases in E. coli starved for thenature amino acids glycine and leucine, respectively (Methods (2005)36:291-298). In vitro, wild-type aminoacyl tRNA synthetases incorporatea wide variety of unnatural amino acids into natural tRNAs asdemonstrated by Hartmann et al. (Proc. Natl. Acad. Sci. (2006)103:4356-4361). In this example, the 20 aminoacyl tRNA synthetases arepurified and incubated in vitro with total tRNA and a variety of naturaland unnatural tRNAs and mass spectrometry is used to determine thestructures of the resulting aa-tRNAs species. Alternatively, geneticscreening methods have been described for developing mutant aminoacyltRNA synthetases in, for example, Saccharomyces cerevisiae that canenzymatically transfer an unnatural amino acid to a specific tRNA(Science (2003) 301:964-967).

Similarly, endogenous E. coli aminoacyl-tRNA synthetases can be used toincorporate unnatural amino acids into an unnatural tRNA such as, forexample, a suppressor tRNA containing a modified anticodon (Chem. Soc.Rev. (2004) 33:422-30).

Unnatural amino acids can also be incorporated into natural and/orunnatural tRNA by, for example, a chemical aminoacylation procedure asdescribed herein and/or known in the art (see, e.g. Nucleic Acids Res.(1989) 17:9649-9660 and Methods (2005) 36:245-251). In general, anyamino acid can be chemically aminoacylated to any tRNA. For example,tRNA^(Phe) can be used to selectively incorporate phenylalanine,p-nitrophenylalanine, or any other amino acid at specific sites in thepolypeptide at a phenylalanine codon. As an example,p-nitrophenylalanine can be chemically aminoacylated to tRNA^(Phe), andcan be incorporated into the polypeptide sequence in place ofphenylalanine. p-nitrophenylalanine can also be arbitrarily added totRNA^(Tyr), for example, and can be incorporated into the polypeptidesequence in place of tyrosine.

Alternatively, an unnatural amino acid can be incorporated into anatural or unnatural tRNA through the use of a peptide nucleic acid(PNA) carrier as described by Sisido et al. (Methods (2005) 36:270-278).In this procedure, a PNA molecule is designed with sequencecomplementary to the 3′ end of the acceptor stock of the tRNA to beaminoacylated. A natural or unnatural amino acid is attached via anamino acid thioester linkage to the PNA. Upon incubation with the tRNA,a PNA/tRNA hybrid is formed. The natural or unnatural amino acid istransferred to the tRNA via an ester exchange and the PNA is released.The aminoacylated tRNA is then separated from the PNA byphenol/chloroform extraction and ethanol precipitation.

Example 7 Incorporation of Natural and Unnatural Amino Acids into aPolypeptide Using an Anti-Stop Codon tRNA

Stop codons in mRNA, represented by UAG, UAA, and UGA, usually signifythe end of the coding sequence and the point at which translation isterminated. However, suppressor tRNA can compete with release factorsand read through the termination signal. Suppressor tRNA have a mutationin the anticodon sequence which allows stop codon recognition. Forexample, a supD mutation in E. coli changes the anticodon sequence oftRNA^(Ser) from CGA to CUA, allowing it to recognize the UAG codon(Nucleic Acids Res. (1983) 11:3823-3832).

The ability of suppressor tRNAs to incorporate amino acids at stopcodons allows for incorporation of up to three unnatural amino acidsinto a single protein using the traditional in vitro translation system(Nucleic Acids Res. (2004) 32:6200-6211).

In the current invention, in which aa-tRNAs are sequentially added andoptionally cleared, multiple stop codons within a coding sequence can beused to arbitrarily incorporate multiple natural and unnatural aminoacids.

Generation of aa-tRNA with Anti-stop Codon

The anticodon of a naturally occurring tRNA, for example tRNA^(Gln), canbe modified to recognize a stop codon by site-directed mutagenesis. AcDNA construct containing the tRNA^(Gln) sequence is generated using,for example, one of the methods described herein. Point mutations in theanticodon are generated using user-defined oligonucleotides of, forexample, 20 bases and a commercially available site-directed mutagenesiskit (e.g. QuikChange® XL, Stratagene, La Jolla, Calif.). The naturalanticodons of tRNA^(Gln) are CUG and UUG at bases 34-36. Mutating, forexample, G36 to A36 of CUG generates an anticodon that corresponds tothe UAG stop codon.

Alternatively, modifications to the anticodon of tRNA^(Gln) can beintroduced using the two oligonucleotide primer strategy of Korencic etal. (Nucleic Acids Res. (2002) 30:105) as described in Example 6. Thelong primer is designed, for example, to include one or more pointmutations that generate anti-stop codons in the anticodon sequence of anatural tRNA sequence.

Suppressor tRNA derived from, for example, tRNA^(Gln), can beaminoacylated with a natural amino acid by either enzymatic or chemicalmethods as described herein or known in the art. Unnatural amino acidsare added to suppressor tRNA by chemical aminoacylation as describedherein or known in the art.

Because of the step-wise addition and optional clearance of aa-tRNAs inthe invention, a single suppressor tRNA can be used for aminoacylationand incorporation of, for example, multiple natural and unnatural aminoacids. Alternatively, the three individual suppressor tRNAs can all beaminoacylated with the same natural or unnatural amino acid.

Generation of Nucleic Acid Template

A nucleic acid template with multiple stop codons can be generated froma cDNA encoding a naturally occurring mRNA using established methods ofsite-directed mutagenesis. For example, a naturally occurring 3-basecodon for phenylalanine UUU is replaced by the stop codon UAG at one ormore sites within the cDNA, maintaining the appropriate reading frame.mRNA with one or more anticodon sites is generated by, for example,transcription run-off as previously described. Alternatively, nucleicacid sequence is generated de novo by a custom commercial service (e.g.Blue Heron Biotechnology, Bothell, Wash.) based on a user-definedsequence. For example, a template sequence can be generated that iscomposed entirely of alternating UAG, UAA, and UGA stop codons.

All three anti-stop codon aa-tRNAs can be charged with all of thecanonical amino acids as well as with unnatural amino acids, dependingupon placement in the amino acid sequence. This would allow, forexample, incorporation of phenylalanine at the UAG, UAA, or UGA codonsusing, for example, Phe-tRNA^(Phe) _(UAG), Phe-tRNA^(Phe) _(UAA), orPhe-tRNA^(Phe) _(UGA), respectively.

Translation Reaction

The nucleic acid template with one or more anticodon sequences is addedto the ribosomal complex and translation initiated. Release factors maybe omitted from the ribosomal complex to prevent premature terminationat the engineered stop codons. Appropriate aa-tRNAs are added andoptionally cleared sequentially from the reaction. When a stop codon istranslocated into the A site of the ribosome, a suppressor aa-tRNA withthe corresponding anti-stop codon is added to the reaction mix. Thesuppressor aa-tRNA incorporates a natural or unnatural amino acid at thesite. Translation is terminated, for example, when the polypeptide ofinterest has been fully synthesized. The end of the coding sequence maycorrespond to one or more stop codons. At this point, release factors,for example RF1, RF2, and RF3, are added to the reaction mix to releasethe translation product from the ribosome.

Example 8 Synthesis of Polypeptides from a Nucleic Acid Template Using aFour-Base Codon/Anticodon Strategy with an Arbitrary aa-tRNA Generationof aa-tRNA with a Four-base Anticodon

Four-base codons on a nucleic acid template can be recognized byaa-tRNAs containing complementary four-base anticodons (e.g. Methods(2005) 36:270-278; Nucleic Acids Res. (2006) 34:1653-1662). User-definedtRNAs are specifically designed to contain an extra base in theanticodon loop, expanding the codon recognition from three to fourbases. For example, the anticodon for phenylalanine is GAA at base pairs34-36 of the human tRNA^(Phe). Addition of an extra base, for example, aG between the As generates a four base anticodon GAGA corresponding tothe codon UCUC.

A modified tRNA with a specified sequence in the anti-codon region canbe generated de novo using the protocols described herein. Analternative scheme for preparing a 4-base anticodon tRNA involvesoverlapping oligonucleotides and primer extension (Methods (2005)36:270-278). A first primer spans half of the desired tRNA sequence andcontains, for example, sequence for the T7 promoter at the 5′end. Asecond reverse primer spans the complementary sequence of the secondhalf of the tRNA with sufficient overlap, for example 20 nucleotides,with the 3′ end of the first oligonucleotide to facilitate annealing ofthe two oligonucleotides. The reverse primer contains the four baseanticodon and variants of the anticodon can be obtained, for example, bymodifying the design of the reverse primer.

Primer extension to generate a double stranded piece of DNA is carriedout using PCR. The reaction mix contains 1 μM each of the two primers,0.2 mM dNTPS and 25 U of Pfu DNA polymerase in 20 mM Tris-HCl (pH 8.8),2 mM MgSO₄, 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% Triton X-100, and 0.1 mg/mlnuclease free bovine serum albumin (BSA). Amplification is carried outusing, for example, 20-30 cycles with a temperature program of 94° C.for 90 s, 55° C. for 2 s, and 72° C. for 30 s. The resulting doublestranded piece of DNA contains a T7 promoter at the 5′ end and the fullsequence of the user-defined tRNA with the 4-base codon. The final tRNAis generated using transcription run-off with T7 polymerase as describedabove.

The resulting tRNA is extracted with phenol and chloroform, precipitatedwith ethanol and further purified by separation on a 10% denaturingpolyacrylamide gel. The RNA on the gel is detected by UV shadowing,excised and extracted from the gel using 2 mM EDTA at room temperatureovernight. After filtration to remove residual gel, the tRNA is againprecipitated with ethanol. The resulting pellet is dissolved in water.Similar procedures have been used to generate tRNA with five-baseanticodons as described by Hohsaka et al. (Nucleic Acids Res. (2001)29:3646-3651)

Incorporation efficiency for different four-base anticodon sequences hasbeen determined using a tRNA library with random mutations in thefour-base anticodon (Biochemistry (2001) 40:11060-11064). Each tRNA isassessed for its ability to incorporate, for example, an unnatural aminoacid such as fluorescently labeled p-nitrophenylalanine into a targettranslation product. A similar approach can be used to change thenucleotides adjacent to the 4-base anticodon in the anticodon loop toimprove efficiency of amino acid incorporation as described by Taira etal. (Nucleic Acids Res. (2006) 34:1653-1662).

Chemical or enzymatic aminoacylation of the resulting tRNA with eithernatural or unnatural amino acids is carried out using the varioustechniques described herein or known in the art.

Nucleic Acid Template

The nucleic acid template for polypeptide synthesis using aa-tRNA with afour-base anticodon can be derived, for example, from modification of anatural mRNA. Site-specific base pair additions in the correspondingcDNA can be accomplished using user-defined primers and site-directedmutagenesis with, for example, a QuikChange® XL Site-DirectedMutagenesis Kit (Stratagene, La Jolla, Calif.) or similar kit from othercommercial sources (e.g. Invitrogen, Carlsbad, Calif.). mRNA isgenerated from the modified cDNA using, for example, T7 polymerase andtranscription run-off as described above. Similar methods can be used togenerate five-base anticodons.

Alternatively, a user-defined nucleic acid template containing, forexample, only 4-base or 5-base codons or a combination thereof with orwith or without 3-base codons can be generated de novo using customcommercial services (e.g. Blue Heron Biotechnology, Bothell, Wash.).

Translation Reaction

The translation reaction is carried out to termination as describedherein with sequential addition of each aa-tRNA, in which the aa-tRNAmay have, for example, a 3-base, 4-base, or 5-base anticodon dependingupon the corresponding nucleic acid template.

Example 9 Synthesis of Polypeptides by Sequential Addition of aa-tRNAsUsing a Mitochondrial Translation System

An in vitro translation system can be isolated from mitochondria as analternative to the cytosolic ribosome complexes described herein.

For example, a reconstructed mitochondrial translation system can beisolated from yeast as described by Pfisterer and Buetow (Proc. Natl.Acad. Sci. (1981) 78:4917-4921). A culture of Saccharomycescarlsbergensis, for example, are grown to high density (OD₆₆₀ 12) andprotoplasts formed by incubation with Glusulase in 1.2 M sorbitol, 10 mMTris maleate (pH 5.7), 1 mM EDTA, and 0.1 M 2-mercaptoethanol for 30minutes at 30° C. followed by centrifugation at 3000×g. The protoplastsare swollen for 20 minutes in 0.6 M sorbitol, 10 mM Tris maleate (pH6.7) 1 mM EDTA, and 0.1% bovine serum albumin (BSA) and lysed using aFrench press at 3000 psi. The lysate is centrifuged for 10 min at 1500×gand the resulting supernatant further centrifuged at 13,000×g for 10minutes. The pellet is washed and resuspended in 50 mM NH4Cl, 10 mM Mgacetate, 10 mM Tris HCl (pH 7.5), and 5 mM 2-mercaptoethanol.Mitochondria are lysed by addition of 5% sodium deoxycholate and rapidpipetting. The lysate is first centrifuged for 10 minutes at 18,000×gand then layered on top of a 1.5 M sucrose gradient and centrifuged for16 hr at 120,000×g. The supernatant is saved and contains componentsnecessary for efficient translation (Proc. Natl. Acad. Sci. (1981)78:4917-4921). Endogenous mitochondrial tRNAs can be removed by runningthe supernatant over a DEAE-cellulose column (Proc. Natl. Acad. Sci.(1981) 78:4917-4921). The ribosome pellet is resuspended in 40 mM TrisHCl (pH 7.8), 10 mM Mg acetate, 30 mM NH4Cl, 5 mM 2-mercaptoethanol, 1mM ATP, 8 mM creatine phosphate with 1.6 μg creatine phosphokinase and20 μM GTP. To this is added the tRNA depleted supernatant in preparationfor translation.

Alternatively, mitochondrial ribosomes can be isolated from rat liversand used for in vitro translation as described, for example, by Ulbrichet al. (Eur. J. Biochem. (1980) 108:337-343. The mitochondrial ribosomesare prepared using a 1.5 M sucrose gradient and the combination ofsupernatant and ribosome pellet are necessary for efficient translation.As with the yeast system described above, endogenous mitochondrial tRNAscan be removed from the mammalian system by running the supernatant overa DEAE-cellulose column.

Aminoacylated tRNAs are generated as described here in. Alternatively,mitochondrial tRNAs can be used in the mitochondrial translation system.There are 22 mitochondrial tRNAs in human and other mammalianmitochondria. The nucleic acid sequence of these tRNAs can be found inpublic databases (e.g. http://mamittrna.u-strasbg.fr/). This sequenceinformation is used to generate natural and unnatural mitochondrial tRNAusing the molecular biology techniques described herein and known in theart. The mitochondrial tRNAs are aminoacylated using the methodsdescribed herein or known in the art.

A natural or user-defined nucleic acid sequence is used as the templatefor translation. Poly(U) mRNA, for example, has been successfullytranslated to polyphenylalanine using in vitro mitochondrial translationsystems from yeast and rat liver (J. Biol. Chem. (1974) 249:6806-6811;Eur. J. Biochem. (1980) 108:337-343; Proc. Natl. Acad. Sci. (1981)78:4917-4721).

Translation proceeds in the reaction chamber using the mitochondrialribosomes and one or more of the methods for sequential combination withnatural or unnatural aa-tRNAs as described herein.

Example 10 Peptide Synthesizing Apparatus

FIG. 24 shows a schematic of an apparatus 1301 (e.g. optionally the sameas apparatus 410 shown in FIG. 1) for biologically synthesizingpeptides, optionally including, for example, a reaction chamber 1300(e.g. optionally the same as one or more peptide synthesizer units 420shown in FIG. 1) connected to one or more reservoirs 2410, 2420, 2430,2440, 2450, 2480 (e.g. optionally the same as one or more sourcing units432 shown in FIG. 1), and where the synthesis is optionally monitored byone or more detectors 2460 (e.g. optionally the same as one or moremonitoring units 440 shown in FIG. 1), and controlled by controlcircuitry (e.g. optionally the same as one or more controller units 422and/or one or more computing units 426 shown in FIG. 1).

The reservoirs 2410, 2420, 2430, 2440, 2450, 2480 optionally include,for example, a reservoir 2410 for ribosomes and initiation components, areservoir 2420 for buffers, a reservoir 2430 for aa-tRNAs, a reservoir2440 for termination components, a reservoir 2450 for waste, and areservoir 2480 for translation product, among others. One or moredetectors 2460 may monitor reactions within the reaction chamber 1300,as well as materials flowing to the waste reservoir 2450 or thetranslation product reservoir 2480, among others. Another part of theapparatus may include control circuitry 2400 for all or part of theprocess for peptide synthesis described herein, and optionally includingthe translation process, for example.

The reaction chamber 1300 may have one or more input ports 2470 toaccommodate addition of reaction components, including components fromone or more reservoirs, and one or more output ports 2490 to eliminatewaste and to recover translation product, for example. The reactionchamber may contain a sensor such as a biosensor or a photodiode.Accordingly, detectors 2460 may include fluorescence detectors, amongothers. The reaction chamber 1300 optionally includes a mechanism forcontrolling fluid temperature, flow rate, and reaction component mixing.Fluids and/or reaction components are moved through the system, forexample, via diffusion, capillary action, centrifugal force,electromotive force, magnetic field, or a pump, among others.

The reaction chamber 1300 may be part of a microchip (see, e.g.microchip 1302 in FIG. 25). Solid substrates for the microchip include,for example, glass (e.g., functionalized glass, a glass slide, poroussilicate glass, a single crystal silicon, quartz, UV-transparentquartz), plastics and polymers (e.g., polystyrene, polypropylene,polyvinylidene difluoride, poly-tetrafluoroethylene, polycarbonate,PDMS, acrylic), metal coated substrates (e.g., gold), siliconsubstrates, latex, membranes (e.g., nitrocellulose, nylon), or a glassslide suitable for surface plasmon resonance (Annu. Rev. Biomed. Eng.(2002) 4:261-286). The surface of the microchip can be modified tofacilitate the stable attachment of linkers, capture probes, or bindingagents, for example. A surface can be amidated by treating the substrateaminosilane, for example. Silane-treated surfaces can be furtherderivatized with homobifunctional and heterobifunctional linkers. Thesubstrate can be derivatized, for example, so it has a hydroxy, an aminoor carboxyl group, N-hydroxy-succinimidyl ester, photoactivatable group,sulfhydryl, ketone, or other functional group available for reaction(see, e.g. U.S. Pat. No. 6,846,638, US 2005/0260653B1).

The reaction chamber 1300 may be a single chamber with a square,rectangular, circular or oval surface configuration, for example (see,e.g. FIG. 25, FIG. 28). The reaction chamber 1300 may also include aseries of channels, for example, in a linear, serpentine, or spiralconfiguration (see, e.g. FIG. 29, FIG. 30, FIG. 31, FIG. 32, FIG. 33,FIG. 35), or it may have a central channel with one or more sidechannels (see, e.g. FIG. 34, FIG. 36, FIG. 37). In some configurations,the reaction chamber may be composed of multiple layers of chambersand/or channels, connected by additional channels, allowing componentsand/or fluids to move on multiple planes (see, e.g. FIG. 32, FIG. 38).Although typically described herein in the singular, reaction chambers1300 may be run in multiples depending on the desired throughput andconnected serially or in parallel, for example. Reaction chambers 1300my also be scaled up and/or scaled down depending on the desiredimplementation.

Chambers and/or channels may be fabricated into the solid substrate ofthe reaction chamber using methods known in the art, for example,micromachining, lithography, embossing, in situ construction, injectionmolding and laser ablation (see, e.g. Annu. Rev. Biomed. Eng. (2002)4:261-286). Alternatively, elastomeric nanochannels may be used, forexample (see, e.g. Nature Materials (2007) 6:424-428).

FIG. 25, FIG. 26, FIG. 27, and FIG. 36 show schematic representations ofillustrative configurations of at least part of one or more apparatus410 for biologically synthesizing peptides.

FIG. 25 shows a schematic of an illustrative configuration of at leastpart of an apparatus 1301 in which a microchip 1302 optionally containsan array of aa-tRNA reservoirs 2430 connected to a reaction chamber 1300by a series of microfabricated channels 2432. In one configuration, forexample, individual aa-tRNA reservoirs 2431 are aligned in a grid withchannels 2432 converging on a central channel 2433 which inputs into thereaction chamber 1300. A wash reservoir 2420 may also converge on thiscentral input channel 2433 and may be used to flush the central channelas well as the reaction chamber 1300 after each aa-tRNA addition, forexample. Waste from each cycle exits through a separate output channelto a waste reservoir 2450. Ribosomes are added via an injection port2470 and the final translation product exits through an output port2480. The reaction chamber 1300 may have optional size exclusionmembranes 2491 that allow outflow of deacylated tRNA and excess aa-tRNA,for example, but not the ribosomal complexes. The translation outflowport 2480 may have an optional affinity chromatography matrix 2481, forexample nickel or cobalt, that allows for purification of thetranslation product as described herein and/or known in the art. Anoptional channel 2421 provides buffers or other components to thechromatography matrix.

FIG. 26 shows a schematic of an illustrative configuration of at leastpart of an apparatus 1303 in which the aa-tRNAs are sourced from areservoir 2430 external to an optional microchip 1302 containing thereaction chamber 1300, and optionally including one or more reservoirssuch as, but not limited to, reservoir 2450 for waste and reservoir 2420for buffers. In one configuration, the aa-tRNA reservoir 2430 is, forexample, a multiwell plate 2434.

In some configurations, the multiwell plate 2434 is directly andoptionally fixedly connected to an input channel 2433 on the reactionchamber 1300 through a main channel 2437 that is fed by channels 2432running from each aa-tRNA reservoir 2431. In some configurations, thechannels 2432 running from each aa-tRNA reservoir 2431 are directlyconnected and optionally fixedly connected to the reaction chamber 1300(not shown).

In some configurations, the reaction chamber 1300 may move relative tothe multiwell plate 2434 and/or the multiwell plate 2434 may moverelative to the reaction chamber 1300 (not shown). In this case, each ofthe channels 2432 running from each aa-tRNA reservoir 2431 include anoptional output port (not shown) that can be aligned with the inputchannel 2433 on the reaction chamber 1300, as appropriate, by movementof either or both of the reaction chamber 1300 and the multiwell plate2434.

FIG. 27 shows a schematic of an illustrative configuration of at leastpart of an apparatus 1304 in which the aa-tRNAs are sourced from areservoir 2430 external to an optional microchip 1302 containing thereaction chamber 1300, and optionally including one or more reservoirssuch as, but not limited to, reservoir 2450 for waste and reservoir 2420for buffers. In one configuration, the aa-tRNA reservoir 2430 is, forexample, a disk 2435. In some configurations, the reservoirs 2431containing aa-tRNAs are optionally arrayed on the perimeter of the disc2435. The disc 2435 is optionally rotated back and forth around a fixedcentral core 2436 to align the appropriate aa-tRNA reservoir 2431 withan input channel 2433 of the reaction chamber 1300.

FIG. 28, FIG. 29, and FIG. 30 show schematic representations ofillustrative configurations of reaction chambers 1300 that areoptionally part of one or more apparatus 410 for biologicallysynthesizing peptides. Although the reaction chambers 1300 areillustratively described for use in one or more of the methods describedherein in which the ribosomes 5310 are fixed in space while the aa-tRNAs5320 are free flowing, they are optionally useful for one or more of theother methods described herein as well.

Ribosomes can be attached to a surface, for example, via abiotin/streptavidin interaction as described herein and/or known in theart (see, e.g. Biosensors & Bioelectronics (2001) 16:745-755). Forexample, a silicon surface of the reaction chamber 1300 may be treatedwith an aminosilane reagent, such as 3-aminopropyltriethoxysilane usingstandard procedures (e.g. Tech Tip #5, Pierce, Rockford, Ill.). Thesilanized silica is subsequently coated with sulfo-NHS-biotin asdescribed herein and/or known in the art. Streptavidin is then added asthe final layer. In one configuration, the ribosomes, biotinylated viathe associated mRNA, are affixed by injection into the input channel2433 and binding to the streptavidin modified surface of the reactionchamber 1300.

FIG. 28 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 composed of a single chamber1310. Ribosomes 5310 are attached to the surface of the chamber 1310optionally after initiation via a streptavidin/biotin interaction. Theaa-tRNAs 5320 are added sequentially, incubated with the ribosomes 5310for a defined or variable interval as determined using methods describedherein, including, for example, as determined by control circuitry 2400.After incubation, the deacylated tRNAs 5330, among other components, arewashed from the reaction chamber 1300.

FIG. 29 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 in which ribosomes 5310 areattached to the surface of a continuous serpentine channel 1320. Theaa-tRNAs 5320 enter one end of the channel and pass by the immobilizedribosomes 5310. The continuous channel may be a variety of shapesincluding, for example, linear or spiral, among others.

FIG. 30 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 designed for protein synthesisusing ribosomes 5310 fixed to beads 5340, for example, and fixed inspace by size exclusion. The reaction chamber 1300 may be constructedfrom, for example, a quartz glass (fused quartz, fused silica) plateinto which a channel 1330 has been cut with an intervening dam 1331 thatallows for fluid flow but limits particle flow, as described in Sato etal. (Anal. Chem. (2000) 72:1144-1147). Beads 5340 with attachedribosomes 5310 as well as free aa-tRNA 5320 are injected into one sideof the channel 1330 and as fluid flows through the channel, the beadsbecome restricted at the dam 1331, while the deacylated tRNA 5330 passbeyond the dam 1331. Reversing the fluid flow allows the used beads tobe flushed from the system.

The reaction chamber 1300 may be composed of three quartz glass plates(cover, middle, bottom) with thicknesses, for example, of 170 μm, 100 μmand 1 mm respectively. Two access holes of 0.5 mm diameter for an inletand an outlet are bored on the cover glass. Two deep channels, forexample, in line with one another and separated by, for example, 3 mmare etched into the middle plate with a CO₂ laser beam. The middle plateis attached to the bottom plate by fusing the optically-smooth surfacesat 1150° C., creating a two part reaction chamber 100 μm in depthseparated by a 3 mm thick dam. Additional etching is used to shave 10 μmoff the top of the dam, allowing for flow of fluid between the tworeaction chambers. A similar microchip device can be made usingphotolithography and wet etching as described by Sato et al. (Lab. Chip.(2004) 4:570-575). Small microspheres precoated, for example, withavidin, streptavidin, or biotin are commercially available (e.g.Luminex, Pierce, Polyscience Inc.). Ribosomes are attached to themicrospheres, for example, using a biotinylated mRNA as described hereinand/or known in the art.

FIG. 31 and FIG. 32 show schematic representations of illustrativeconfigurations of reaction chambers 1300 that are optionally part of oneor more apparatus 410 for biologically synthesizing peptides. Althoughthe reaction chambers 1300 are illustratively described for use in oneor more of the methods described herein in which the aa-tRNAs 5320 aretethered to a surface while the ribosomes 5310 are free flowing, theyare optionally useful for one or more of the other methods describedherein as well.

FIG. 31 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 in which the aa-tRNAs 5320 areattached to the surface of a channel 1320 with a serpentineconfiguration. Ribosome complexes 5310 pass through the channel 1320,interacting with the tethered aa-tRNA 5320. The aa-tRNA 5320 areoptionally tethered in the order required for target peptide synthesis.

FIG. 32 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 in which the specific aa-tRNAs5320 are tethered in discrete wells 2437 of a multiwell plate 1340. Anupper chamber 1341 above the multiwell plate 1340 has a grid of channels1342 with openings 1343 down to each discrete well 2437. The ribosomes5310 are attached to magnetic beads, for example, as described hereinand/or known in the art. The beads are moved along the channels 1342 ofthe grid by magnetic force. The beads are directed to the next aa-tRNAin the target peptide sequence optionally using control circuitry 2400,and dropped into the well. After a defined or variable time interval, asdescribed herein, magnetic force may be used to lift the beads out ofthe well and to move the beads along the grid to the next appropriatewell. Tethered aa-tRNAs may be replenished, for example, by swapping outexhausted wells 2437. In some configurations, the specific aa-tRNAs 5320are free in solution in each discrete well 2437, and are replenished byadding more aa-tRNA 5320 to the well from an optional externalreservoir.

FIG. 33, FIG. 34, FIG. 35, FIG. 36, FIG. 37, and FIG. 38 show schematicrepresentations of illustrative configurations of reaction chambers 1300that are optionally part of one or more apparatus 410 for biologicallysynthesizing peptides. Although the reaction chambers 1300 areillustratively described for use in one or more of the methods describedherein in which the ribosomes 5310 and the aa-tRNAs 5320 are both freein solution, encapsulated in aqueous bubbles, or attached to movablemicrospheres, the reaction chambers are optionally useful for one ormore of the other methods described herein as well. The reaction chamber1300 may be a continuous channel optionally linear, serpentine, square,and/or spiral (among others) in configuration, optionally with multipleside channels through which ribosomes 5310 and/or aa-tRNAs 5320 flow.

FIG. 33 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 at four different stages duringan illustrative round of peptide synthesis. The reaction chamber 1300includes a channel 1350 optionally configured in a square (or, forexample, an oval or circle (not shown)). In the reaction chamber 1300,an input port 1351 allows for addition of reagents and components,including aa-tRNA 5320. The output port 1353 allows for removal ofwaste, deacylated tRNAs 5330, and retrieval of the translation product,among other components.

The numbered diagrams (1, 2, 3, 4) indicate the progressive movement ofribosomes 5310 and aa-tRNA 5320 during a round of peptide synthesis. InPart 1, ribosomes 5310, either free or attached to beads, for example,are loaded into the channel 1350. Specific aa-tRNAs 5320 are added in anappropriate sequence through the input port 1351, and the ribosomes 5310and aa-tRNAs 5320 flow through a mixer 1352 consisting of obstructionsto the flow of buffer. In Part 2, the ribosomes 5310 and aa-tRNAs 5320travel along the channel 1350 for a specified or variable time intervaldefined by methods described herein optionally including using controlcircuitry 2400. In Part 3, the ribosomes 5310 with associated nascentpolypeptide chain and deacylated tRNAs 5330 reach the output port 1353.The ribosomes are excluded from passage through the output port 1353 bysize exclusion, either because of the size of the ribosome complex 5310alone or because of the size of an associated bead, for example. Thedeacylated tRNAs 5330 pass out of the reaction chamber 1300 through theoutput port 1353. In Part 4, a valve 1354 is opened which allows theribosomes 5310 to return to the start of the channel, whereupon the nextaa-tRNAs 5320 are encountered. The cycle is repeated until translationis complete.

FIG. 34 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 in which the main channel 1360is linear with multiple side channels 1361. The number of side channels1361 may be higher or lower than the 29 shown depending on the targetpeptide to be sequenced, among other considerations. The channel isoptionally looped, so that the ribosomes 5310 pass the same sidechannels 1361 multiple times.

Each side channel 1361 is optionally sourced with one type of aa-tRNA5320 at a given time. The aa-tRNAs 5320 are optionally loaded into themultiple side channels in the same sequence as the desired codingsequence, or are optionally loaded in a set sequence, but then releasedin the correct order as the ribosomes 5310 approach. The ribosomecomplex 5310, preinitiated with nucleic acid is injected into one end ofthe continuous channel. The complex flows through the main channel 1360,spending a defined or variable interval optionally determined by controlcircuitry 2400 at appropriate side channels 1361 in association withappropriate aa-tRNA 5320.

FIG. 35 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 in which the reaction chamber1300 is a disc 1370 (e.g. a CD) into which a spiral channel 1371 hasbeen etched. Ribosomes 5310 are loaded into the channel 1371 at acentral input 1372. The ribosomes 5310 move down the channel by, forexample, centrifugal force. Multiple side channels 1374 are optionallypre-loaded with aa-tRNA 5320 in a target sequence or are optionallyattached to multiple sources of aa-tRNA 5320 that can optionally bereleased according to control-circuitry 2400. As the ribosomes move pastthe side channels 1374 aa-tRNA 5320 are optionally released in a targetsequence for incorporation into the nascent polypeptide which may beaccessed at an outlet port 1373 at the termination of the spiral.

FIG. 36 shows a schematic representation of an illustrativeconfiguration of at least part of one or more apparatus 410 forbiologically synthesizing peptides, including a reaction chamber 1300and a reservoir 2430. The reaction chamber 1300 is a circular channel1380 with multiple side channels 1381. Each side channel 1381 optionallycontains one type of aa-tRNA at a time. The ribosome complexes 5310 areadded to the channel at input 1382. The ribosome complexes 5310 movefrom one side channel 1381 to the next. In one illustrativeconfiguration, for example, there are 25 side channels 1381 (shown). Aseach aa-tRNA is added via a side channel 1381 to the reaction chamber1300, the side channel is refilled from, for example, an externalreservoir 2430 with the aa-tRNA that will be needed 25 steps in thefuture. The waste and translation products, for example, are collectedfrom an output 1383.

FIG. 37 shows a schematic representation of an illustrativeconfiguration of a reaction chamber 1300 in which the reaction chamber1300 is composed of a main linear channel 1390 which overlaps with aserpentine channel 1391. The aa-tRNAs 5320 move in a continuous streamalong channel 1390. The ribosomes 5310 move in a serpentine pattern inchannel 1391, crossing through the flow of aa-tRNAs at the junction 1392of the two channels. The ribosomes 5310 and/or aa-tRNAs 5320 may be freein solution, encapsulated in an aqueous bubble, or affixed tomicrospheres. The ribosomes 5310 and aa-tRNAs 5320 optionally move inopposite directions, such that the ribosomes 5310 pick up eachsuccessive aa-tRNA 5320 in an order calculated to achieve the targetprotein sequence. A portion of the channel 1393 traversed by theribosome 5310 is optionally coated with a peptide nucleic acid (PNA),for example, that recognizes and binds deacylated t-RNA 5330.

FIG. 38 shows a schematic representation of an illustrativeconfiguration of a multilayer reaction chamber 1300 in which ribosomesand aa-tRNAs may be free flowing. In one configuration, the ribosomes5311, 5312, 5313 and aa-tRNAs 5321, 5322, 5323 move in parallel streamsalong a square channel 1350 (optionally oval or circular, among others(not shown)). There are optionally multiple stacked channels 1350. Theribosomes flow into each channel 1350 via input 1355 and pass to anotherchannel through output 1356. Similarly aa-tRNAs flow into each channel1350 via input 1357 and pass to another channel through output 1358.Each set of ribosome complexes 5311, 5312, and 5313 and aa-tRNAs 5321,5322, and 5323 are labeled, for example, with a unique fluorescent dye.There may be a single population of ribosomes initiated with a uniquecoding sequence. Alternatively, there may be a mixed population ofribosomes initiated with more than one coding sequence, with each codingsequence/ribosome type having a unique fluorescence identifier.Similarly, each aa-tRNA is optionally labeled with a unique fluorescentdye. Detectors 2460 monitor the fluorescence. When the appropriatecombinations of ribosome-associated and aa-tRNA-associated fluorescenceare detected in proximity to one another, the two streams are pushedtogether by the lowering of a partial barrier, for example, to allow forinteraction between the ribosome and the aa-tRNA. In one configuration,the ribosomes and/or the aa-tRNAs are affixed to magnetic beads andmagnetic force is used to push the two components together. For example,magnetic microspheres of varied size and fluorescence are commerciallyavailable (Luminex, Austin, Tex.) and can be used in combination withfluorescence monitoring to detect proximity of appropriate ribosome andaa-tRNA pair.

In one aspect, the disclosure is drawn to one or more methods comprisingreceiving a first input associated with a first possible dataset, thefirst possible dataset including data representative of one or morecharged tRNA sequences; and determining temporal-spatial parameters forsynthesizing one or more peptides based on a first possible data set.One or more of these methods may be used as part of one or more methodsof target peptide synthesis and/or implemented on one or more apparatus410 for target peptide synthesis.

FIG. 7 shows an operational flow 100 representing illustrativeembodiments of operations related to determining temporal-spatialparameters for synthesizing one or more peptides based on a firstpossible dataset. In FIG. 7, and in the following figures that includevarious illustrative embodiments of operational flows, discussion andexplanation may be provided with respect to apparatus and methodsdescribed herein, and/or with respect to other examples and contexts.The operational flows may also be executed in a variety of othercontexts and environments, and or in modified versions of thosedescribed herein. In addition, although some of the operational flowsare presented in sequence, the various operations may be performed invarious repetitions, concurrently, and/or in other orders than thosethat are illustrated.

After a start operation, the operational flow 100 moves to a receivingoperation 110 where receiving a first input may be associated with afirst possible dataset, the first possible dataset including datarepresentative of one or more charged tRNA sequences. For example, afirst input may include data representative of a target peptidesequence, a target nucleic acid sequence, a target biological assembler,and/or target biological assembler components. A first input may alsoinclude data representative of the identity and sequence of chargedtRNA.

An optional accessing operation 210 accesses the first possible datasetin response to the first input. For example, data representative of atarget peptide sequence, a target nucleic acid sequence, a targetbiological assembler, and/or target biological assembler components maybe accessed. Data representative of the identity and sequence of chargedtRNA may also be accessed.

An optional generating operation 310 generates the first possibledataset in response to the first input. For example, data representativeof a target peptide sequence, a target nucleic acid sequence, a targetbiological assembler, and/or target biological assembler components maybe generated. Data representative of the identity and sequence ofcharged tRNA may also be generated.

An optional determining operation 410 determines a graphicalillustration of the first possible dataset. For example, datarepresentative of a target peptide sequence, a target nucleic acidsequence, a target biological assembler, and/or target biologicalassembler components may be graphically represented. Data representativeof the identity and sequence of charged tRNA may also be graphicallyrepresented.

Then, a determining operation 510, determines temporal-spatialparameters for synthesizing one or more peptides based on the firstpossible dataset. For example, data representative of temporal-spatialparameters for synthesizing one or more peptides based on a targetpeptide sequence, a target nucleic acid sequence, a target biologicalassembler, and/or target biological assembler components may bedetermined. Data representative of temporal-spatial parameters forsynthesizing one or more peptides based on the sequence of charged tRNAmay also be determined.

Operations 110 to 510 may be performed with respect to a digitalrepresentation (e.g. digital data) of, for example, data representativeof a target peptide sequence, a target nucleic acid sequence, a targetbiological assembler, and/or target biological assembler components. Thelogic may accept a digital or analog (for conversion into digital)representation of an input and/or provide a digitally-encodedrepresentation of a graphical illustration, where the input may beimplemented and/or accessed locally or remotely.

Operations 110 to 510 may be performed related to either a local or aremote storage of the digital data, or to another type of transmissionof the digital data. In addition to inputting, accessing querying,recalling, calculating, determining or otherwise obtaining the digitaldata, operations may be performed related to storing, assigning,associating, displaying or otherwise archiving the digital data to amemory, including for example, sending and/or receiving a transmissionof the digital data from a remote memory. Accordingly, any suchoperations may involve elements including at least an operator (e.g.human or computer) directing the operation, a transmitting computer,and/or receiving computer, and should be understood to occur in theUnited States as long as at least one of these elements resides in theUnited States.

FIG. 8 illustrates optional embodiments of the operational flow 100 ofFIG. 7. FIG. 8 shows illustrative embodiments of the receiving operation110, receiving a first input associated with a first possible dataset,the first possible dataset including data representative of one or morecharged tRNA sequences, including operations receiving types of inputsand data entry and may include at least one additional operation.Receiving operations may optionally include, but are not limited to,operation 1100, operation 1101, operation 1102, operation 1103,operation 1104, and/or operation 1105.

At the optional operation 1100, the first input may include one or moreof a target or one or more target components. At the optional operation1101, a first data entry associated with a first possible dataset may bereceived. At the optional operation 1105, a first data entry at leastpartially identifying one or more elements of the first possible datasetmay be received.

At the optional operation 1102, a first data entry associated with afirst possible dataset may be received that may include one or more of atarget or one or more target components. A first data entry associatedwith a first possible dataset may be received that may include at leastpartially identifying one or more of a target structure, a peptidesequence, a nucleic acid sequence, a biological assembler, one or morebiological assembler components, one or more amino acids, one or morecharged tRNA, or one or more tRNA. A first data entry associated with afirst possible dataset may be received that may include receiving afirst data entry at least partially identifying one or more of one ormore domains of a target structure, one or more shapes of the targetstructure, one or more charges of a target structure, one or morefunctions of a target structure, an mRNA sequence, a RNA sequence, acDNA sequence, a DNA sequence, one or more natural amino acids, one ormore unnatural amino acids, one or more tRNA charged with natural aminoacids, one or more tRNA charged with unnatural amino acids, one or moretRNA charged with arbitrary amino acids, one or more natural tRNA, oneor more unnatural tRNA, one or more anti-stop codon tRNA, one or moreanti-singlet codon tRNA, one or more anti-doublet codon tRNA, one ormore anti-triplet codon tRNA, one or more anti-quadruplet codon tRNA,one or more anti-quintuplet codon tRNA, or one or more anti-sextupletcodon tRNA.

At the optional operation 1103, a first data entry may be received froma graphical user interface, or at the optional operation 1104, from atleast one submission element of a graphical user interface.

FIG. 9 illustrates optional embodiments of the operational flow 100 ofFIG. 7. FIG. 9 shows illustrative embodiments of the optional accessingoperation 210, accessing the first possible dataset in response to thefirst input, including operations accessing the first possible datasetand may include at least one additional operation. Accessing operationsmay optionally include, but are not limited to, operation 2100,operation 2101, operation 2102, operation 2103, operation 2104,operation 2105, operation 2106, operation 2107, operation 2108,operation 2109, operation 2110, operation 2111, and operation 2112.

At the optional operation 2100, a first possible dataset may be accessedin response to a first input, the first input including one or more of atarget or one or more target components. At the optional operation 2101,a first possible dataset may be accessed from within a first databaseassociated with a plurality of targets and target components. At theoptional operation 2102, a first possible dataset may be accessed byassociating one or more of a target and/or one or more target componentswith one or more elements of the first possible dataset. At the optionaloperation 2103, a first possible dataset may be accessed using adatabase management system engine that is configured to query a firstdatabase to retrieve a first possible dataset therefrom. At the optionaloperation 2104, a first possible dataset may be accessed bycorresponding one or more of a target and one or more target componentswith one or more elements of a first possible dataset.

A first possible dataset may be accessed by associating one or more of atarget, one or more of a target structure, a peptide sequence, a nucleicacid sequence, a biological assembler, one or more biological assemblercomponents, one or more amino acids, one or more charged tRNA, or one ormore tRNA with one or more elements of the first possible dataset. Afirst possible dataset may be accessed by associating one or more of oneor more domains of a target structure, one or more shapes of a targetstructure, one or more charges of a target structure, one or morefunctions of a target structure, an mRNA sequence, a RNA sequence, acDNA sequence, a DNA sequence, one or more natural amino acids, one ormore unnatural amino acids, one or more tRNA charged with natural aminoacids, one or more tRNA charged with unnatural amino acids, one or moretRNA charged with arbitrary amino acids, one or more natural tRNA, oneor more unnatural tRNA, one or more anti-stop codon tRNA, one or moreanti-singlet codon tRNA, one or more anti-doublet codon tRNA, one ormore anti-triplet codon tRNA, one or more anti-quadruplet codon tRNA,one or more anti-quintuplet codon tRNA, or one or more anti-sextupletcodon tRNA with one or more elements of the first possible dataset. Afirst possible dataset may be accessed as being associated with one ormore of a target or one or more target components, based on one or morecharacterizations stored in association with one or more elements of thefirst possible dataset and related to one or more of a peptide sequence,a nucleic acid sequence, biological assembler, one or more biologicalassembler components, one or more amino acids, one or more charged tRNA,or one or more tRNA.

At the optional operation 2105, a first request associated with a firstpossible dataset may be received. At the optional operation 2106, afirst request associated with a first possible dataset may be received,the first request selecting one or more of a target or one or moretarget components. At the optional operation 2107, a first request froma graphical user interface may be received. At the optional operation2108, a first request from at least one submission element of agraphical user interface may be received. At the optional operation2109, a first request from at least one submission element of agraphical user interface may be received, one or more first requests atleast partially identifying one or more elements of a first possibledataset. At the optional operation 2110, a first request from at leastone submission element of a graphical user interface may be received,one or more first requests at least partially selecting one or moreelements of a first possible dataset. At the optional operation 2111, afirst request from at least one submission element of a graphical userinterface may be received, one or more first requests providinginstructions identifying one or more of a target or one or more targetcomponents.

A first request from at least one submission element of a graphical userinterface may be received, one or more first requests providinginstructions identifying a target structure, a peptide sequence, anucleic acid sequence, a biological assembler, one or more biologicalassembler components, one or more amino acids, one or more charged tRNA,or one or more tRNA. A first request from at least one submissionelement of a graphical user interface may be received, one or more firstrequests providing instructions identifying one or more domains of atarget structure, one or more shapes of the target structure, one ormore charges of a target structure, one or more functions of a targetstructure, an MRNA sequence, a RNA sequence, a cDNA sequence, a DNAsequence, one or more natural amino acids, one or more unnatural aminoacids, one or more tRNA charged with natural amino acids, one or moretRNA charged with unnatural amino acids, one or more tRNA charged witharbitrary amino acids, one or more natural tRNA, one or more unnaturaltRNA, one or more anti-stop codon tRNA, one or more anti-singlet codontRNA, one or more anti-doublet codon tRNA, one or more anti-tripletcodon tRNA, one or more anti-quadruplet codon tRNA, one or moreanti-quintuplet codon tRNA, or one or more anti-sextuplet codon tRNA.

At the optional operation 2112, a first possible dataset may be accessedin response to a first request, the first request specifying one or moreof a target or one or more target components and at least one otherinstruction.

FIG. 10 illustrates optional embodiments of the operational flow 100 ofFIG. 7. FIG. 10 shows illustrative embodiments of the optionalgenerating operation 310, generating the first possible dataset inresponse to the first input, including operations generating the firstpossible dataset and may include at least one additional operation.Generating operations may optionally include, but are not limited to,operation 3100, operation 3101, operation 3102, operation 3103,operation 3104, operation 3105, operation 3106; operation 3107,operation 3108, operation 3109, operation 3110, operation 3111,operation 3112, operation 3113, operation 3114, operation 3115.

At the optional operation 3100, a first possible dataset may begenerated in response to a first input, the first input including one ormore of a target or one or more target components. At the optionaloperation 3101, a first possible dataset may be generated from withinthe first database associated with a plurality of targets and targetcomponents. At the optional operation 3102, a first possible dataset maybe generated by associating one or more of a target and one or moretarget component with one or more elements of the first possibledataset. At the optional operation 3103, a first possible dataset may begenerated using a database management system engine that is configuredto query a first database to retrieve a first possible datasettherefrom. At the optional operation 3104, a first possible dataset maybe generated by corresponding one or more of a target and one or moretarget components with one or more elements of the first possibledataset.

A first possible dataset may be generated by associating one or more ofthe target structure, at peptide sequence, a nucleic acid sequence, abiological assembler, one or more biological assembler components, oneor more amino acids, one or more charged tRNA, or one or more tRNA, withone or more elements of the first possible dataset. A first possibledataset may be generated by associating one or more of one or moredomains of the target structure, one or more shapes of the targetstructure, one or more charges of the target structure, one or morefunctions of the target structure, an mRNA sequence, a RNA sequence, acDNA sequence, a DNA sequence, one or more natural amino acids, one ormore unnatural amino acids, one or more tRNA charged with natural aminoacids, one or more tRNA charged with unnatural amino acids, one or moretRNA charged with arbitrary amino acids, one or more natural tRNA, oneor more unnatural tRNA, one or more anti-stop codon tRNA, one or moreanti-singlet codon tRNA, one or more anti-doublet codon tRNA, one ormore anti-triplet codon tRNA, one or more anti-quadruplet codon tRNA,one or more anti-quintuplet codon tRNA, or one or more anti-sextupletcodon tRNA, with one or more elements of the first possible dataset.

At the optional operation 3105, a first request associated with a firstpossible dataset may be received. At the optional operation 3106, afirst request associated with the first possible dataset may bereceived, the first request selecting one or more of a target or one ormore target components. At the optional operation 3107, a first requestfrom a graphical user interface may be received. At the optionaloperation 3108, a first request may be received from at least onesubmission element of a graphical user interface. At the optionaloperation 3109, a first request may be received from at least onesubmission element of a graphical user interface, the first request atleast partially identifying one or more elements of the first possibledataset. At the optional operation 3110, a first request may be receivedfrom at least one submission element of a graphical user interface, thefirst request selecting one or more elements of the first possibledataset. At the optional operation 3111, a first request may be receivedfrom at least one submission element of the graphical user interface,the first request providing instructions identifying one or more of atarget or one or more target components.

A first request may be received from at least one submission element ofthe graphical user interface, the first request providing instructionsidentifying the target structure, a peptide sequence, a nucleic acidsequence, a biological assembler, one or more biological assemblercomponents, one or more amino acids, one or more charged tRNA, or one ormore tRNA. A first request may be received from at least one submissionelement of the graphical user interface, the first request providinginstructions identifying one or more domains of the target structure,one or more shapes of the target structure, one or more charges of thestructure, one or more functions of a target structure, an mRNAsequence, a RNA sequence, a cDNA sequence, a DNA sequence, one or morenatural amino acids, one or more unnatural amino acids, one or more tRNAcharged with natural amino acids, one or more tRNA charged withunnatural amino acids, one or more tRNA charged with arbitrary aminoacids, one or more natural tRNA, one or more unnatural tRNA, one or moreanti-stop codon tRNA, one or more anti-singlet codon tRNA, one or moreanti-doublet codon tRNA, one or more anti-triplet codon tRNA, one ormore anti-quadruplet codon tRNA, one or more anti-quintuplet codon tRNA,or one or more anti-sextuplet codon tRNA.

At the optional operation 3112, a first request associated with thefirst possible dataset may be received, and at the optional operation3113, the first possible dataset may be generated in response to thefirst request, the first request specifying one or more of a target orone or more target components and at least one other instruction. At theoptional operation 3114, a first request may be received, the firstrequest specifying one or more of a target or one or more targetcomponents, and at the optional operation 3115, the first possibledataset may be generated in response to the first request at leastpartially by performing an analysis of the one or more of a target orone or more target components.

FIG. 11 illustrates optional embodiments of the operational flow 100 ofFIG. 7. FIG. 11 shows illustrative embodiments of the optionaldetermining operation 410, determining a graphical illustration of thefirst possible dataset, including operations determining a graphicalillustration of the first possible dataset and may include at least oneadditional operation. Determining operations may optionally include, butare not limited to, operation 4100, operation 4101, operation 4102,operation 4103, operation 4104, operation 4105, operation 4106,operation 4107, and operation 4108.

At the optional operation 4100, a graphical illustration for inclusionin a display element of a graphical user interface may be determined. Atthe optional operation 4101, an analysis of one or more elements of thefirst possible dataset may be performed to determine a first possibleoutcome, and at the optional operation 4102 the graphical illustrationmay be determined based on the analysis. At the optional operation 4103,analysis of one or more elements of the first possible dataset may beperformed to determine a first possible outcome, the first possibleoutcome including one or more of a possible risk, a possible result, ora possible consequence; and at the optional operation 4104 the graphicalillustration may be determined based on the analysis. At the optionaloperation 4105, an analysis of one or more elements of the firstpossible dataset may be performed to determine the first possibleoutcome, the first possible outcome including one or more of a possiblerisk, a possible result, or a possible consequence, and at the optionaloperation 4106, the graphical illustration may be determined includingone or more of a target or one or more target components in associationwith a visual indicator related to the first possible outcome. At theoptional operation 4107, correlation between the first possible outcomeand a type or characteristic of a visual indicator used in the graphicalillustration to represent the first possible outcome may be determined.At the optional operation 4108, the graphical illustration of the firstpossible outcome of use of one or more of the target structure, apeptide sequence, a nucleic acid sequence, a biological assembler, oneor more biological assembler components, one or more amino acids, one ormore charged tRNA, one or more tRNA, or one or more tRNA chargingcomponents may be determined. The graphical illustration of a firstpossible outcome of use of one or more of one or more domains of thetarget structure, one or more shapes of the target structure, one ormore charges of the structure, one or more functions of a targetstructure, an mRNA sequence, a RNA sequence, a cDNA sequence, a DNAsequence, one or more natural amino acids, one or more unnatural aminoacids, one or more tRNA charged with natural amino acids, one or moretRNA charged with unnatural amino acids, one or more tRNA charged witharbitrary amino acids, one or more natural tRNA, one or more unnaturaltRNA, one or more anti-stop codon tRNA, one or more anti-singlet codontRNA, one or more anti-doublet codon tRNA, one or more anti-tripletcodon tRNA, one or more anti-quadruplet codon tRNA, one or moreanti-quintuplet codon tRNA, or one or more anti-sextuplet codon tRNA maybe determined.

FIG. 12 illustrates optional embodiments of the operational flow 100 ofFIG. 7. FIG. 12 shows illustrative embodiments of the determiningoperation 510, determining temporal-spatial parameters for synthesizingone or more target peptides based on the first possible dataset,including operations determining temporal-spatial parameters forimplementing the first possible dataset and may include at least oneadditional operation. Determining operations may optionally include, butare not limited to, operation 5100, operation 5101, operation 5102,operation 5103, operation 5104, and operation 5105.

At the optional operation 5100, temporal-spatial parameters forsynthesizing one or more peptides based on the first possible datasetmay be determined, the first possible dataset including one or more of atarget or one or more target components. At the optional operation 5101,an analysis of one or more elements of the first possible dataset may beperformed, and at the optional operation 5102, the temporal spatialparameters for providing one or more target components may bedetermined, based on the analysis. At the optional operation 5103, ananalysis of one or more elements of the first possible dataset and atleast one additional instruction may be performed, and at the optionaloperation 5104, the temporal-spatial parameters for providing one ormore target components and/or removing one or more target components maybe determined, based on the analysis. At the optional operation 5105,the temporal-spatial parameters for providing one or more targetcomponents and/or removing one or more target components may bedetermined, the temporal-spatial parameters including one or more oftiming, sequence, or location.

FIG. 13 shows a schematic of a partial view of an illustrative computerprogram product 1800 that includes a computer program for executing acomputer process on a computing device. An illustrative embodiment ofthe example computer program product is provided using a signal bearingmedium 1802, and may include at least one instruction of 1804: one ormore instructions for receiving a first input associated with a firstpossible dataset, the first possible dataset including datarepresentative of one or more charged tRNA sequence; one or moreinstructions for accessing the first possible dataset in response to thefirst input; one or more instructions for generating the first possibledataset in response to the first input; one or more instructions fordetermining a graphical illustration of the first possible dataset; orone or more instructions for determining temporal-spatial parameters forsynthesizing one or more peptides based on the first possible dataset.The one or more instructions may be, for example, computer executableand/or logic implemented instructions. In some embodiments, the signalbearing medium 1802 of the one or more computer program 1800 productsinclude a computer readable medium 1806, a recordable medium 1808,and/or a communications medium 1810.

FIG. 14 shows a schematic of an illustrative system 1900 in whichembodiments may be implemented. The system 1900 may include a computingsystem environment. The system 1900 also illustrates aresearcher/scientist/investigator/operator 104 using a device 1904, thatis optionally shown as being in communication with a computing device1902 by way of an optional coupling 1906. The optional coupling mayrepresent a local, wide area, or peer-to-peer network, or may representa bus that is internal to a computing device (e.g. in illustrativeembodiments the computing device 1902 is contained in whole or in partwithin the device 1904 or within one or more apparatus 410, or one ormore computing units 426, or one or more controller units 422, or one ormore monitoring units 440). An optional storage medium 1908 may be anycomputer storage medium.

The computing device 1902 includes one or more computer executableinstructions 1910 that when executed on the computing device 1902 causethe computing device 1902 to receive the first input associated with thefirst possible dataset, the first possible dataset including datarepresentative of one or more charged tRNA sequences; optionally accessthe first possible dataset in response to the first input; optionallygenerate the first possible dataset in response the first input;optionally determine a graphical illustration of the first possibledataset; and determine temporal-spatial parameters for synthesizing oneor more peptides at least partially based on a first possible dataset.In some illustrative embodiments, the computing device 1902 mayoptionally be contained in whole or in part within an apparatus 410and/or one or more peptide synthesizer units 420 of FIG. 1 (e.g. one ormore computing units 426, and/or one or more controller units 422,and/or one or more monitoring units 440), or may optionally be containedin whole or in part within the researcher device 1904.

The system 1900 includes at least one computing device (e.g. 1904 and/or1902 and/or one or more computing units 426 of FIG. 1) on which thecomputer-executable instructions 1910 may be executed. For example, oneor more of the computing devices (e.g. 1902, 1904, 426) may execute theone or more computer executable instructions 1910 and output a resultand/or receive information from the researcher (optionally from one ormore monitoring unit 440) on the same or a different computing device(e.g. 1902, 1904, 426) and/or output a result and/or receive informationfrom one or more peptide synthesizer units 420 and/or one or moremonitoring units 440 and/or one or more computing units 426 and/or oneor more controller units 422 in order to perform and/or implement one ormore of the techniques, processes, or methods described herein, or othertechniques.

The computing device (e.g. 1902 and/or 1904 and/or 426) may include oneor more of a desktop computer, a workstation computer, a computingsystem comprised a cluster of processors, a networked computer, a tabletpersonal computer, a laptop computer, or a personal digital assistant,or any other suitable computing unit or may be part of any one of theapparatus 410 described herein. In some embodiments, an apparatus 410,one or more peptide synthesizer units 420 and/or one or more monitoringunits 440 and/or one or more controller units 422 may be operable tocommunicate with any one of the one or more computing devices (e.g. 1902and/or 1904 and/or 426) that may be operable to communicate with adatabase to access the first possible dataset and/or subsequentdatasets. In some embodiments, the computing device (e.g. 1902 and/or1904 and/or 426) is operable to communicate with the peptide biologicalsynthesis apparatus 410.

In one aspect, the disclosure is drawn to one or more methods comprisingreceiving a first input associated with a first possible dataset, thefirst possible dataset including data representative of one or moreaspects of target peptide synthesis; and determining temporal-spatialparameters for sequentially co-localizing one or more target componentsbased on the first possible dataset and/or for separating one or moretarget components based on the first possible dataset. One or more ofthese methods may be used as part of one or more methods of targetpeptide synthesis and/or implemented on one or more apparatus 410 fortarget peptide synthesis.

FIG. 15 shows an operational flow 600 representing illustrativeembodiments of operations related to determining temporal-spatialparameters for sequentially co-localizing one or more target componentsbased on the first possible dataset and/or for separating one or moretarget components based on the first possible dataset. In FIG. 15, andin the following figures that includes various illustrative embodimentsof operational flows, discussion and explanation may be provided withrespect to apparatus and methods described herein, and/or with respectto other examples and contexts. The operational flows may also beexecuted in a variety of other contexts and environments, and or inmodified versions of those described herein. In addition, although someof the operational flows are presented in sequence, the variousoperations may be performed in various repetitions, concurrently, and/orin other orders than those that are illustrated. The operational flowsmay be performed in real time during target peptide synthesis.

After a start operation, the operational flow 600 moves to a receivingoperation 610 where a first input may be associated with a firstpossible dataset, the first possible dataset including datarepresentative of one or more aspects of target synthesis. For example,a first input may include data representative of the progress of targetpeptide synthesis. Data representative of the progress of target peptidesynthesis may be manually or automatically gathered, or derived frommonitoring the progress of target peptide synthesis. Monitoring may beoptionally performed by one or more monitoring units 440.

An optional accessing operation 710 accesses the first possible datasetin response to the first input. For example, data representative of theprogress of target peptide synthesis may be accessed. Such data may bemanually or automatically generated, or derived from one or moremonitoring units 440.

An optional generating operation 810 generates the first possibledataset in response to the first input. For example, data representativeof the progress of target peptide synthesis may be generated. Such datamay be manually or automatically generated, or derived from one or moremonitoring units 440.

An optional determining operation 910 determines a graphicalillustration of the first possible dataset. For example, datarepresentative of the progress of target peptide synthesis may begraphically represented. Such data may be manually or automaticallygenerated, or derived from one or more monitoring units 440.

Then, a determining operation 1010, determines temporal-spatialparameters for optionally sequentially co-localizing one or more targetcomponents based on the first possible dataset and/or for separating oneor more target components based on the first possible dataset. Forexample, temporal-spatial parameters for sequentially co-localizing oneor more target components based on the first possible dataset and/or forseparating one or more target components based on the first possibledataset may be determined at least partially based on datarepresentative of the progress of target peptide synthesis. Such datamay be manually or automatically generated, or derived from one or moremonitoring units 440.

Operations 610 to 1010 may be performed with respect to a digitalrepresentation (e.g. digital data) of, for example, data representativeof progress of a target peptide synthesis. The logic may accept adigital or analog (for conversion into digital) representation of aninput and/or provide a digitally-encoded representation of a graphicalillustration, where the input may be implemented and/or accessed locallyor remotely.

Operations 610 to 1010 may be performed related to either a local or aremote storage of the digital data, or to another type of transmissionof the digital data. In addition to inputting, accessing querying,recalling, calculating, determining or otherwise obtaining the digitaldata, operations may be performed related to storing, assigning,associating, displaying or otherwise archiving the digital data to amemory, including for example, sending and/or receiving a transmissionof the digital data from a remote memory. Accordingly, any suchoperations may involve elements including at least an operator (e.g.human or computer) directing the operation, a transmitting computer,and/or receiving computer, and should be understood to occur in theUnited States as long as at least one of these elements resides in theUnited States.

FIG. 16 illustrates optional embodiments of the operational flow 600 ofFIG. 15. FIG. 16 shows illustrative embodiments of the receivingoperation 610, including operations receiving a first input, optionallydata entry, associated with a first possible dataset, the first inputincluding data representative of one or more aspects of targetsynthesis, and optionally one or more additional operations. Receivingoperations may optionally include, but are not limited to, operation6100, operation 6101, operation 6102, operation 6103, operation 6104,operation 6105, operation 6106, operation 6107, operation 6108,operation 6109, operation 6110, and/or operation 6111.

At the optional operation 6100, the first input may include datarepresentative of amino acid incorporation into one or more peptides,biological assembler activity, nucleic acid translocation, and/or tRNArelease. At the optional operation 6101, the first input may includedata representative of availability of one or more nucleic acid codons,concentrations of one or more charged tRNA and/or one or more tRNA,presence or absence of one or more charged tRNA and/or one or more tRNA,and/or presence or absence of one or more anti-codons on one or morecharged tRNA and/or one or more tRNA.

At the optional operation 6110, the first input may be associated withmonitoring amino acid incorporation, biological assembly activity,nucleic acid translocation, and/or tRNA release. At the optionaloperation 6111, the first input may be associated with monitoringavailability of one or more nucleic acid codons, concentrations of oneor more charged tRNA and/or one or more tRNA, presence or absence of oneor more charged tRNA and/or one or more tRNA, and/or presence or absenceof one or more anti-codons on one or more charged tRNA and/or one ormore tRNA. Monitoring may be performed by one or more monitoring units440.

At the optional operation 6102, a first data entry associated with thefirst possible dataset may be received. At the optional operation 6109,a first data entry at least partially identifying one or more elementsof the first possible dataset may be received.

At the optional operation 6103, a first data entry associated with thefirst possible dataset may be received that may include receiving thefirst input associated with amino acid incorporation, biologicalassembly activity, nucleic acid translocation, and/or tRNA release. Atthe optional operation 6104, a first data entry associated with thefirst possible dataset may be received that may include datarepresentative of availability of one or more nucleic acid codons,concentrations of one or more charged tRNA and/or one or more tRNA,presence or absence of one or more charged tRNA and/or one or more tRNA,and/or presence or absence of one or more anti-codons on one or morecharged tRNA and/or one or more tRNA.

At the optional operation 6105, a first data entry from one or morepeptide synthesizer units 420 may be received. At the optional operation6106, a first data entry from one or more monitoring units 440, one ormore computing units 426 and/or one or more controller units 422 may bereceived.

At the optional operation 6107, a first data entry may be received froma graphical user interface, or at the optional operation 6108 from atleast one submission element of a graphical user interface.

FIG. 17 illustrates optional embodiments of the operational flow 600 ofFIG. 15. FIG. 17 shows illustrative embodiments of the optionalaccessing operation 710, including operations accessing the firstpossible dataset in response to the first input, and may include atleast one additional operation. Accessing operations may optionallyinclude, but are not limited to, operation 7100, operation 7101,operation 7102, operation 7103, operation 7104, operation 7105,operation 7106, operation 7107, operation 7108, operation 7109,operation 7110, and/or operation 7111.

At the optional operation 7100, a first possible dataset may be accessedin response to a first input, the first input including datarepresentative of amino acid incorporation into one or more peptides,biological assembler activity, nucleic acid translocation, and/or tRNArelease. At the optional operation 7101, a first possible dataset may beaccessed in response to a first input, the first input including datarepresentative of availability of one or more nucleic acid codons,concentrations of one or more charged tRNA and/or one or more tRNA,presence or absence of one or more charged tRNA and/or one or more tRNA,and/or presence or absence of one or more anti-codons on one or morecharged tRNA and/or one or more tRNA.

At the optional operation 7102, a first possible dataset may be accessedby associating data representative of amino acid incorporation into oneor more peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release with one or more elements of thefirst possible dataset. At the optional operation 7103, a first possibledataset may be accessed by associating data representative ofavailability of one or more nucleic acid codons, concentrations of oneor more charged tRNA and/or one or more tRNA, presence or absence of oneor more charged tRNA and/or one or more tRNA, and/or presence or absenceof one or more anti-codons on one or more charged tRNA and/or one ormore tRNA with one or more elements of the first possible dataset.

At the optional operation 7104, a first possible dataset may be accessedby corresponding data representative of amino acid incorporation intoone or more peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release with one or more elements of thefirst possible dataset. At the optional operation 7105, a first possibledataset may be accessed by corresponding data representative ofavailability of one or more nucleic acid codons, concentrations of oneor more charged tRNA and/or one or more tRNA, presence or absence of oneor more charged tRNA and/or one or more tRNA, and/or presence or absenceof one or more anti-codons on one or more charged tRNA and/or one ormore tRNA with one or more elements of the first possible dataset.

At the optional operation 7106, a first possible dataset may be accessedin response to the first input at least partially based on receiving afirst request associated with the first possible dataset. At theoptional operation 7107, a first possible dataset may be accessed inresponse to the first input at least partially based on receiving afirst request associated with the first possible dataset, the firstrequest selecting one or more target components. At the optionaloperation 7108, a first possible dataset may be accessed in response tothe first input at least partially based on receiving a first requestfrom a graphical user interface, or the optional operation 7109,receiving a first request from at least one submission element of agraphical user interface. At the optional operation 7110, a firstpossible dataset may be accessed in response to the first input at leastpartially based on receiving a first request from at least onesubmission element of a graphical user interface, the first requestproviding instructions identifying one or more target components. At theoptional operation 7111, a first possible dataset may be accessed inresponse to the first input at least partially based on receiving afirst request from at least one submission element of a graphical userinterface, the first request specifying one or more target componentsand at least one other instruction.

FIG. 18 and FIG. 19 illustrate optional embodiments of the operationalflow 600 of FIG. 15. FIG. 18 and FIG. 19 show illustrative embodimentsof the optional generating operation 810, including operationsgenerating the first possible dataset in response to the first input,and may include at least one additional operation. Generating operationsmay optionally include, but are not limited to, operation 8100,operation 8101, operation 8102, operation 8103, operation 8104,operation 8105, operation 8108, operation 8109, operation 8110,operation 8111, operation 8112, operation 8113, operation 8114,operation 8115, operation 8116 and/or operation 8117.

At the optional operation 8100, the first possible dataset is generatedin response to the first input, the first input including datarepresentative of amino acid incorporation into one or more peptides,biological assembler activity, nucleic acid translocation, and/or tRNArelease. At the optional operation 8101, the first possible dataset isgenerated in response to the first input, the first input including datarepresentative of availability of one or more nucleic acid codons,concentrations of one or more charged tRNA and/or one or more tRNA,presence or absence of one or more charged tRNA and/or one or more tRNA,and/or presence or absence of one or more anti-codons on one or morecharged tRNA and/or one or more tRNA.

At the optional operation 8102, the first possible dataset is generatedin response to the first input by associating data representative ofamino acid incorporation into one or more peptides, biological assembleractivity, nucleic acid translocation, and/or tRNA release with one ormore elements of the first possible dataset. At the optional operation8103, the first possible dataset is generated in response to the firstinput by associating data representative of availability of one or morenucleic acid codons, concentrations of one or more charged tRNA and/orone or more tRNA, presence or absence of one or more charged tRNA and/orone or more tRNA, and/or presence or absence of one or more anti-codonson one or more charged tRNA and/or one or more tRNA with one or moreelements of the first possible dataset.

At the optional operation 8104, the first possible dataset is generatedin response to the first input by corresponding data representative ofamino acid incorporation into one or more peptides, biological assembleractivity, nucleic acid translocation, and/or tRNA release with one ormore elements of the first possible dataset. At the optional operation8105, the first possible dataset is generated by corresponding datarepresentative of one or more of availability of one or more nucleicacid codons, concentrations of one or more charged tRNA and/or one ormore tRNA, presence or absence of one or more charged tRNA and/or one ormore tRNA, and/or presence or absence of one or more anti-codons on oneor more charged tRNA and/or one or more tRNA with one or more elementsof the first possible dataset.

At the optional operation 8108, the first possible dataset is generatedin response to the first input at least partially by performing ananalysis of data representative of amino acid incorporation into one ormore peptides, biological assembler activity, nucleic acidtranslocation, and/or tRNA release. At the optional operation 8109, thefirst possible dataset is generated in response to the first input atleast partially by performing an analysis of data representative of oneor more of availability of one or more nucleic acid codons,concentrations of one or more charged tRNA and/or one or more tRNA,presence or absence of one or more charged tRNA and/or one or more tRNA,and/or presence or absence of one or more anti-codons on one or morecharged tRNA and/or one or more tRNA.

In some embodiments, the first possible dataset is generated in responseto the first input, wherein receiving a first input associated with afirst possible dataset comprises receiving a first request associatedwith the first possible dataset 8110. In some embodiments, the firstpossible dataset is generated in response to the first input, whereinreceiving a first input associated with a first possible datasetcomprises receiving a first request associated with the first possibledataset, the first request selecting one or more target components 8111.In some embodiments, the first possible dataset is generated in responseto the first input, wherein receiving a first input associated with afirst possible dataset comprises receiving a first request from agraphical user interface 8112. In some embodiments, the first possibledataset is generated in response to the first input, wherein receiving afirst input associated with a first possible dataset comprises receivinga first request from at least one submission element of a graphical userinterface 8113. In some embodiments, the first possible dataset isgenerated in response to the first input, wherein receiving a firstinput associated with a first possible dataset comprises receiving afirst request from at least one submission element of a graphical userinterface, the first request providing instructions identifying one ormore target components 8114. In some embodiments, the first possibledataset is generated in response to the first input, wherein receiving afirst input associated with a first possible dataset comprises receivinga first request from at least one submission element of a graphical userinterface, the first request specifying one or more target componentsand at least one other instruction 8115. In some embodiments, the firstpossible dataset is generated in response to the first input, whereinreceiving a first input associated with a first possible datasetcomprises receiving a first request associated with the first possibledataset 8116; and generating the first possible dataset in response tothe first request, the first request specifying one or more targetcomponents and at least one other instruction 8117.

FIG. 20 illustrates optional embodiments of the operational flow 600 ofFIG. 15. FIG. 20 shows illustrative embodiments of the optionaldetermining operation 910, including operations determining a graphicalillustration of the first possible dataset, and may include at least oneadditional operation. Determining operations may optionally include, butare not limited to, operation 9100, operation 9101, operation 9102,operation 9103, operation 9104, operation 9105, operation 9106, and/oroperation 9107.

At the optional operation 9100, a graphical illustration of the firstpossible dataset is determined for inclusion in a display element of agraphical user interface. At the optional operation 9107, a graphicalillustration of the first possible dataset is determined by determininga correlation between a first possible outcome and a type orcharacteristic of a visual indicator used in the graphical illustrationto represent the first possible outcome.

In some embodiments, a graphical illustration of the first possibledataset is determined by performing an analysis of one or more elementsof the first possible dataset to determine a first possible outcome9101; and determining the graphical illustration, based on the analysis9102. In some embodiments, a graphical illustration of the firstpossible dataset is determined by performing an analysis of one or moreelements of the first possible dataset to determine a first possibleoutcome, the first possible outcome including one or more of a currentstatus or a time to completion 9103; and determining the graphicalillustration, based on the analysis 9104. In some embodiments, agraphical illustration of the first possible dataset is determined byperforming an analysis of one or more elements of the first possibledataset to determine a first possible outcome, the first possibleoutcome including one or more of a current status or a time tocompletion 9105; and determining the graphical illustration includingone or more of a target or one or more target components in associationwith a visual indicator related to the first possible outcome 9106.

FIG. 21 illustrates optional embodiments of the operational flow 600 ofFIG. 15. FIG. 21 shows illustrative embodiments of the determiningoperation 1010, including operations determining temporal-spatialparameters for optionally sequentially co-localizing one or more targetcomponents based on the first possible dataset or optionallysequentially separating one or more target components based on the firstpossible dataset, and may include at least one additional operation.Determining operations may optionally include, but are not limited to,operation 10100, operation 10101, operation 10102, operation 10103,operation 10104, operation 10105, and/or operation 10106.

At the optional operation 10100, temporal-spatial parameters aredetermined for optionally sequentially co-localizing the one or moretarget components based on the first possible dataset, and/or optionallysequentially separating the one or more target components based on thefirst possible dataset, the first possible dataset including datarepresentative of one or more of amino acid incorporation into one ormore peptides, biological assembler activity, nucleic acidtranslocation, or tRNA release. At the optional operation 10101,temporal-spatial parameters are determined for optionally sequentiallyco-localizing the one or more target components based on the firstpossible dataset, and/or optionally sequentially separating the one ormore target components based on the first possible dataset, the firstpossible dataset including data representative of availability of one ormore nucleic acid codons, concentrations of one or more charged tRNAand/or one or more tRNA, presence or absence of one or more charged tRNAand/or one or more tRNA, and/or presence or absence of one or moreanti-codons on one or more charged tRNA and/or one or more tRNA. At theoptional operation 10106, temporal-spatial parameters are determined foroptionally sequentially co-localizing the one or more target componentsbased on the first possible dataset, and/or optionally sequentiallyseparating the one or more target components based on the first possibledataset, the temporal-spatial parameters including timing, sequence,location, and/or order.

In some embodiments, temporal-spatial parameters are determined foroptionally sequentially co-localizing one or more target componentsbased on the first possible dataset, and/or optionally sequentiallyseparating one or more target components based on the first possibledataset by performing an analysis of one or more elements of the firstpossible dataset 10102; and determining the temporal-spatial parametersfor optionally sequentially co-localizing the one or more targetcomponents based on the first possible dataset, and/or optionallysequentially separating the one or more target components based on thefirst possible dataset, based on the analysis 10103. In someembodiments, temporal-spatial parameters are determined for optionallysequentially co-localizing one or more target components based on thefirst possible dataset, and/or optionally sequentially separating one ormore target components based on the first possible dataset by performingan analysis of one or more elements of the first possible dataset and atleast one additional instruction 10104; and determining thetemporal-spatial parameters for optionally sequentially co-localizingthe one or more target components based on the first possible dataset,and/or optionally sequentially separating the one or more targetcomponents based on the first possible dataset, based on the analysis10105.

FIG. 22 shows a schematic of a partial view of an illustrative computerprogram product 2200 that includes a computer program for executing acomputer process on a computing device. An illustrative embodiment ofthe example computer program product is provided using a signal bearingmedium 2202, and may include at least one instruction of 2204: one ormore instructions for receiving a first input associated with a firstpossible dataset, the first possible dataset including datarepresentative of one or more aspects of target peptide synthesis; oneor more instructions for accessing the first possible dataset inresponse to the first input; one or more instructions for generating thefirst possible dataset in response to the first input; one or moreinstructions for determining a graphical illustration of the firstpossible dataset; and/or one or more instructions for determiningtemporal-spatial parameters for one or more of sequentiallyco-localizing one or more target components based on the first possibledataset or optionally sequentially separating one or more targetcomponents based on the first possible dataset. The one or moreinstructions may be, for example, computer executable and/or logicimplemented instructions. In some embodiments, the signal bearing medium2202 of the one or more computer program products 2200 include acomputer readable medium 2206, a recordable medium 2208, and/or acommunications medium 2210.

FIG. 23 shows a schematic of an illustrative system 2300 in whichembodiments may be implemented. The system 2300 may include a computingsystem environment. The system 2300 also illustrates aresearcher/scientist/investigator/operator 104 using a device 2304, thatis optionally shown as being in communication with a computing device2302 by way of an optional coupling 2306. The optional coupling mayrepresent a local, wide area, or peer-to-peer network, or may representa bus that is internal to a computing device (e.g. in illustrativeembodiments the computing device 2302 is contained in whole or in partwithin the device 2304 or within one or more apparatus 410, or one ormore computing units 426, or one or more controller units 422, or one ormore monitoring units 440). An optional storage medium 2308 may be anycomputer storage medium.

The computing device 2302 includes one or more computer executableinstructions 2310 that when executed on the computing device 2302 causethe computing device 2302 to receive a first input associated with afirst possible dataset, the first possible dataset including datarepresentative of one or more aspects of target peptide synthesis;optionally access the first possible dataset in response to the firstinput; optionally generate the first possible dataset in response to thefirst input; optionally determine a graphical illustration of the firstpossible dataset; and determine temporal-spatial parameters for one ormore of optionally sequentially co-localizing one or more targetcomponents based on the first possible dataset or optionallysequentially separating one or more target components based on the firstpossible dataset. In some illustrative embodiments, the computing device2302 may optionally be contained in whole or in part within one or moreunits of an apparatus 410 of FIG. 1 (e.g. one or more computing units426 and/or one or more controller units 422 and/or one or moremonitoring units 440), or may optionally be contained in whole or inpart within the researcher device 2304.

The system 2300 includes at least one computing device (e.g. 2304 and/or2302 and/or one or more computing units 426 of FIG. 1) on which thecomputer-executable instructions 2310 may be executed. For example, oneor more of the computing devices (e.g. 2302, 2304, 426) may execute theone or more computer executable instructions 2310 and output a resultand/or receive information from the researcher (optionally from one ormore monitoring unit 440) on the same or a different computing device(e.g. 2302, 2304, 426) and/or output a result and/or receive informationfrom an apparatus 410, one or more peptide synthesizer units 420, one ormore controller units 422, and/or one or more monitoring units 440 inorder to perform and/or implement one or more of the techniques,processes, or methods described herein, or other techniques.

The computing device (e.g. 2302 and/or 2304 and/or 426) may include oneor more of a desktop computer, a workstation computer, a computingsystem comprised a cluster of processors, a networked computer, a tabletpersonal computer, a laptop computer, or a personal digital assistant,or any other suitable computing unit. In some embodiments, one or morepeptide synthesis units 420 and/or one or more monitoring units 440 maybe operable to communicate with any one of the computing devices (e.g.2302 and/or 2304 and/or 426) that may be operable to communicate with adatabase to access the first possible dataset and/or subsequentdatasets. In some embodiments, the computing device (e.g. 2302 and/or2304 and/or 426) is operable to communicate with the peptide biologicalsynthesizer apparatus 410.

There is little distinction left between hardware and softwareimplementations of aspects of systems; the use of hardware or softwareis generally (but not always, in that in certain contexts the choicebetween hardware and software can become significant) a design choicerepresenting cost vs. efficiency tradeoffs. There are various vehiclesby which processes and/or systems and/or other technologies describedherein can be effected (e.g., hardware, software, and/or firmware), andthat the preferred vehicle will vary with the context in which theprocesses and/or systems and/or other technologies are deployed. Forexample, if an implementer determines that speed and accuracy areparamount, the implementer may opt for a mainly hardware and/or firmwarevehicle; if flexibility is paramount, the implementer may opt for amainly software implementation; or, yet again alternatively, theimplementer may opt for some combination of hardware, software, and/orfirmware.

The foregoing detailed description has set forth various embodiments ofthe devices and/or processes via the use of block diagrams, flowcharts,and/or examples. Insofar as such block diagrams, flowcharts, and/orexamples contain one or more functions and/or operations, it will beunderstood by those within the art that each function and/or operationwithin such block diagrams, flowcharts, or examples can be implemented,individually and/or collectively, by a wide range of hardware, software,firmware, or virtually any combination thereof. In one embodiment,several portions of the subject matter described herein may beimplemented via Application Specific Integrated Circuits (ASICs), FieldProgrammable Gate Arrays (FPGAs), digital signal processors (DSPs), orother integrated formats. However, those skilled in the art willrecognize that some aspects of the embodiments disclosed herein, inwhole or in part, can be equivalently implemented in integratedcircuits, as one or more computer programs running on one or morecomputers (e.g., as one or more programs running on one or more computersystems), as one or more programs running on one or more processors(e.g., as one or more programs running on one or more microprocessors),as firmware, or as virtually any combination thereof, and that designingthe circuitry and/or writing the code for the software and or firmwarewould be well within the skill of one of skill in the art in light ofthis disclosure. In addition, those skilled in the art will appreciatethat the mechanisms of the subject matter described herein are capableof being distributed as a program product in a variety of forms, andthat an illustrative embodiment of the subject matter described hereinapplies regardless of the particular type of signal bearing medium usedto actually carry out the distribution. Examples of a signal bearingmedium include, but are not limited to, the following: a recordable typemedium such as a floppy disk, a hard disk drive, a Compact Disc (CD), aDigital Video Disk (DVD), a digital tape, a computer memory, etc.; and atransmission type medium such as a digital and/or an analogcommunication medium (e.g., a fiber optic cable, a waveguide, a wiredcommunications link, a wireless communication link, etc.).

Those skilled in the art will recognize that it is common within the artto describe devices and/or processes in the fashion set forth herein,and thereafter use engineering practices to integrate such describeddevices and/or processes into data processing systems. That is, at leasta portion of the devices and/or processes described herein can beintegrated into a data processing system via a reasonable amount ofexperimentation. Those having skill in the art will recognize that atypical data processing system generally includes one or more of asystem unit housing, a video display device, a memory such as volatileand non-volatile memory, processors such as microprocessors and digitalsignal processors, computational entities such as operating systems,drivers, graphical user interfaces, and applications programs, one ormore interaction devices, such as a touch pad or screen, and/or controlsystems including feedback loops and control motors (e.g., feedback forsensing position and/or velocity; control motors for moving and/oradjusting components and/or quantities). A typical data processingsystem may be implemented utilizing any suitable commercially availablecomponents, such as those typically found in datacomputing/communication and/or network computing/communication systems.

The herein described subject matter sometimes illustrates differentcomponents contained within, or connected with, different othercomponents. It is to be understood that such depicted architectures aremerely exemplary, and that in fact many other architectures can beimplemented which achieve the same functionality. In a conceptual sense,any arrangement of components to achieve the same functionality iseffectively “associated” such that the desired functionality isachieved. Hence, any two components herein combined to achieve aparticular functionality can be seen as “associated with” each othersuch that the desired functionality is achieved, irrespective ofarchitectures or intermedial components. Likewise, any two components soassociated can also be viewed as being “operably connected”, or“operably coupled”, to each other to achieve the desired functionality,and any two components capable of being so associated can also be viewedas being “operably couplable”, to each other to achieve the desiredfunctionality. Specific examples of operably couplable include but arenot limited to physically mateable and/or physically interactingcomponents and/or wirelessly interactable and/or wirelessly interactingcomponents and/or logically interacting and/or logically interactablecomponents.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

For ease of reading, all values described herein, and all numericalranges described herein are approximate and should be read as includingthe word “about” or “approximately” prior to each numeral, unlesscontext indicates otherwise. For example, the range “0.0001 to 0.01” ismeant to read as “about 0.0001 to about 0.01.”

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to inventions containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

All references, including but not limited to patents, patentapplications, and non-patent literature are hereby incorporated byreference herein in their entirety.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1. A method comprising: flowing one or more ribosome-based assemblers inone or more channels into a reaction chamber of a peptide synthesisapparatus, wherein the reaction chamber includes two or more chargedtRNA tethered therein in a selected spatial order related to a targetpeptide synthesis sequence; and co-localizing sequentially the one ormore ribosome-based assemblers with the two or more charged tRNA in thereaction chamber.
 2. The method of claim 1, wherein co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA in the reaction chamber comprises: synthesizing atarget peptide by co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA.
 3. Themethod of claim 1, wherein two or more of the two or more charged tRNAhave the same anti-codon and are optionally charged with different aminoacids.
 4. The method of claim 1, wherein two or more of the two or morecharged tRNA have optionally one or more of different anti-codons ordifferent tRNA and are charged with the same amino acid.
 5. The methodof claim 1, further comprising: co-localizing the at least two of thetwo or more charged tRNA at the one or more different locations.
 6. Themethod of claim 5, wherein co-localizing the at least two or morecharged tRNA at the one or more different locations comprises:co-localizing the at least two of the two or more charged tRNA at theone or more different locations at one or more first identifiable timeintervals.
 7. The method of claim 1, wherein co-localizing sequentiallythe one or more biological assemblers with the two or more charged tRNAin the reaction chamber_occurs at least partially following theco-localizing the at least two of the two or more charged tRNA at theone or more different locations.
 8. The method of claim 1, wherein eachof the two or more charged tRNA are charged with a natural amino acid,an unnatural amino acid, or an arbitrary amino acid.
 9. The method ofclaim 8, further comprising: charging two or more tRNA each with thenatural amino acid, the arbitrary amino acid, or the one unnatural aminoacid.
 10. The method of claim 1, wherein the two or more charged tRNAoptionally include one or more charged anti-stop codon tRNA.
 11. Themethod of claim 1, further comprising: selecting the two or more chargedtRNA.
 12. The method of claim 11, wherein selecting the two or morecharged tRNA comprises: selecting the two or more charged tRNA based atleast partially on a target peptide sequence.
 13. The method of claim11, wherein selecting the two or more charged tRNA comprises: selectingthe two or more charged tRNA based at least partially on a nucleic acidprotein coding sequence.
 14. The method of claim 1, whereinco-localizing sequentially the one or more ribosome-based assemblerswith the two or more charged tRNA in the reaction chamber comprises:co-localizing sequentially the one or more ribosome-based assemblerswith the two or more charged tRNA at one or more second identifiabletime intervals.
 15. The method of claim 14, wherein the one or moresecond identifiable time intervals are at least partially based on oneor more of a predicted rate of incorporation of two or more amino acidsinto one or more peptides, a predicted rate of activity of the one ormore ribosome-based assemblers, a predicted rate of translocation of oneor more nucleic acids, or a predicted rate of release of tRNA.
 16. Themethod of claim 1, further comprising: monitoring one or more of aminoacid incorporation into one or more peptides, ribosome-based assembleractivity, nucleic acid translocation, or tRNA release.
 17. The method ofclaim 16, wherein co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA in thereaction chamber comprises: co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA at one ormore second identifiable time intervals, and wherein the one or moresecond identifiable time intervals are at least partially based on oneor more of the amino acid incorporation into the one or more peptides,the ribosome-based assembler activity, the nucleic acid translocation,or the tRNA release.
 18. The method of claim 16, wherein co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA in the reaction chamber comprises: co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA at one or more second identifiable time intervals, andwherein the one or more second identifiable time intervals are at leastpartially based on availability of one or more nucleic acid codons. 19.The method of claim 1, further comprising: monitoring one or more ofpresence or absence, concentration, or composition of one or more of thetwo or more charged tRNA or one or more tRNA.
 20. The method of claim19, wherein co-localizing sequentially the one or more ribosome-basedassemblers with the two or more charged tRNA in the reaction chambercomprises: co-localizing sequentially the one or more ribosome-basedassemblers with the two or more charged tRNA at one or more secondidentifiable time intervals, and wherein the one or more secondidentifiable time intervals are at least partially based on theconcentrations of one or more of the two or more charged tRNA or the oneor more tRNA.
 21. The method of claim 19, wherein co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA in the reaction chamber comprises: co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA at one or more second identifiable time intervals, andwherein the one or more second identifiable time intervals are at leastpartially based on one or more of presence or absence of one or more ofthe two or more charged tRNA or the one or more tRNA.
 22. The method ofclaim 19, wherein co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA in thereaction chamber comprises: co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA at one ormore second identifiable time intervals, and wherein the one or moresecond identifiable time intervals are at least partially based on oneor more of presence or absence of one or more anti-codons on one or moreof the two or more charged tRNA or the one or more tRNA.
 23. The methodof claim 1, wherein co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA in thereaction chamber comprises: co-localizing the one or more ribosome-basedassemblers with one or more first charged tRNA at one or more firstlocations; co-localizing the one or more ribosome-based assemblers withone or more second charged tRNA at one or more second locations; andoptionally repeating.
 24. The method of claim 1, wherein co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA in the reaction chamber comprises: co-localizing theone or more ribosome-based assemblers with one or more first chargedtRNA at one or more first locations, each of the one or more firstcharged tRNA charged with a first arbitrary amino acid; removing the oneor more ribosome-based assemblers from the one or more first locations;co-localizing the one or more ribosome-based assemblers with one or moresecond charged tRNA at one or more second locations, each of the one ormore second charged tRNA charged with a second arbitrary amino acid;removing the one or more ribosome-based assemblers from the one or moresecond locations; and optionally repeating.
 25. The method of claim 1,wherein co-localizing sequentially the one or more ribosome-basedassemblers with the two or more charged tRNA in the reaction chambercomprises: co-localizing the one or more ribosome-based assemblers withone or more first charged tRNA at one or more first locations, each ofthe one or more first charged tRNA charged with a first arbitrary aminoacid; removing one or more of the one or more first charged tRNA or oneor more first tRNA, the one or more first tRNA released during peptidesynthesis; co-localizing the one or more ribosome-based assemblers withone or more second charged tRNA at one or more second locations, each ofthe one or more second charged tRNA charged with a second arbitraryamino acid; removing one or more of the one or more second charged tRNAor one or more second tRNA, the one or more second tRNA released duringpeptide synthesis; and optionally repeating.
 26. The method of claim 1,further comprising: consuming sequentially the two or more charged tRNA.27. The method of claim 1, further comprising: eliminating one or moreof the two or more charged tRNA or one or more tRNA.
 28. The method ofclaim 1, further comprising: separating the one or more ribosome-basedassemblers from one or more of the two or more charged tRNA or one ormore tRNA.
 29. The method of claim 28, wherein separating the one ormore ribosome-based assemblers from one or more of the two or morecharged tRNA or one or more tRNA comprises: separating sequentially theone or more biological assemblers from one or more of the two or morecharged tRNA or one or more tRNA.
 30. The method of claim 28, whereinseparating the one or more ribosome-based assemblers from one or more ofthe two or more charged tRNA or one or more tRNA comprises: separatingsequentially the one or more ribosome-based assemblers from one or moreof the two or more charged tRNA or one or more tRNA at one or more thirdidentifiable time intervals.
 31. The method of claim 1, whereinco-localizing sequentially the one or more ribosome-based assemblerswith the two or more charged tRNA in the reaction chamber comprises:co-localizing sequentially the one or more ribosome-based assemblerswith the two or more charged tRNA in a defined sequence.
 32. The methodof claim 31, further comprising: determining the defined sequence forco-localizing the one or more ribosome-based assemblers with the two ormore charged tRNA.
 33. The method of claim 32, wherein determining thedefined sequence for co-localizing the one or more ribosome-basedassemblers with the two or more charged tRNA comprises: determining thedefined sequence for co-localizing the one or more ribosome-basedassemblers with the two or more charged tRNA based at least partially ona target peptide sequence.
 34. The method of claim 32, whereindetermining the defined sequence for co-localizing the one or moreribosome-based assemblers with the two or more charged tRNA comprises:determining the defined sequence for co-localizing the one or moreribosome-based assemblers with the two or more charged tRNA based atleast partially on a nucleic acid protein coding sequence.
 35. Themethod of claim 34, wherein a protein coding region of the nucleic acidsequence includes two or more stop codons.
 36. The method of claim 35,wherein the protein coding region includes at least three stop codons.37. The method of claim 35, wherein the protein coding region includestwo or more alternating stop codons.
 38. The method of claim 35, whereinthe protein coding region includes one or more of one or more singletcodons, one or more doublet codons, one or more triplet codons, one ormore quadruplet codons or one or more quintuplet codons.
 39. The methodof claim 1, wherein co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA in thereaction chamber comprises: co-localizing sequentially one or morepeptide assemblers with the two or more charged tRNA.
 40. The method ofclaim 1, wherein co-localizing sequentially the one or moreribosome-based assemblers with the two or more charged tRNA in thereaction chamber comprises: co-localizing sequentially one or moreprokaryotic ribosome-based assemblers with the two or more charged tRNA.41. The method of claim 1, wherein co-localizing sequentially the one ormore ribosome-based assemblers with the two or more charged tRNA in thereaction chamber comprises: co-localizing sequentially one or moreeukaryotic ribosome-based assemblers with the two or more charged tRNA.42. The method of claim 1, wherein co-localizing sequentially the one ormore ribosome-based assemblers with the two or more charged tRNA in thereaction chamber comprises: co-localizing sequentially one or moremitochondrial ribosome-based assemblers with the two or more chargedtRNA.
 43. The method of claim 1, further comprising: selecting the oneor more ribosome-based assemblers.
 44. The method of claim 1, furthercomprising: assembling one or more components of the one or moreribosome-based assemblers.
 45. The method of claim 44, whereinassembling one or more components of the one or more ribosome-basedassemblers comprises: assembling the one or more components of the oneor more ribosome-based assemblers at one or more fourth identifiabletime intervals.
 46. The method of claim 44, further comprising:selecting the one or more components of the one or more ribosome-basedassemblers.
 47. The method of claim 1, wherein co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA in the reaction chamber comprises: co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA, wherein the one or more ribosome-based assemblers areco-localized with one or more nucleic acids.
 48. The method of claim 47,further comprising: synchronizing the co-localizing sequentially the oneor more ribosome-based assemblers with the two or more charged tRNA,with a protein coding sequence of the one or more nucleic acids.
 49. Themethod of claim 1, further comprising: co-localizing one or more nucleicacids with the one or more ribosome-based assemblers.
 50. The method ofclaim 49, wherein co-localizing one or more nucleic acids with the oneor more ribosome-based assemblers comprises: co-localizing the one ormore nucleic acids with the one or more ribosome-based assemblers at oneor more fifth identifiable time intervals.
 51. The method of claim 49,wherein the co-localizing sequentially the one or more ribosome-basedassemblers with the two or more charged tRNA occurs at least partiallyfollowing the co-localizing the one or more nucleic acids with the oneor more ribosome-based assemblers.
 52. The method of claim 49, furthercomprising: selecting the one or more nucleic acids.
 53. The method ofclaim 52, further comprising: synthesizing the one or more nucleicacids.
 54. A method of extra-cellular peptide synthesis comprising:flowing one or more ribosome-based assemblers in one or more channelsinto a reaction chamber of a computer-controlled peptide synthesisapparatus, wherein the reaction chamber includes two or more chargedtRNA tethered in a selected spatial order related to a target peptidesynthesis sequence; and co-localizing sequentially the one or moreribosome-based assemblers with the-two or more charged tRNA in thereaction chamber.
 55. A method comprising: determining an assemblyorder; flowing one or more ribosome-based assemblers in one or morechannels into a reaction chamber of a peptide synthesis apparatus,wherein the reaction chamber includes two or more charged tRNA tetheredin a selected spatial order related to a target peptide synthesissequence; and co-localizing the one or more ribosome-based assemblerswith two or more charged tRNA based at least partially on the assemblyorder.
 56. The method of claim 1, wherein flowing one or moreribosome-based assemblers in one or more channels into a reactionchamber of a peptide synthesis apparatus includes: flowing the one ormore ribosome-based assemblers from at least one biological assemblerreservoir into the reaction chamber via one or more biological assemblerchannels.
 57. The method of claim 56, wherein the reaction chamber ispart of a microchip.
 58. The method of claim 1, wherein co-localizingsequentially the one or more ribosome-based assemblers with the two ormore charged tRNA in the reaction chamber includes: flowing a pluralityof ribosome-based assemblers from corresponding biological assemblerreservoirs, through corresponding biological assembler channels, througha common input channel, and into the reaction chamber.
 59. The method ofclaim 1, wherein the reaction chamber exhibits a serpentineconfiguration.
 60. The method of claim 54, wherein the reaction chamberexhibits a serpentine configuration.
 61. The method of claim 55, whereinthe reaction chamber exhibits a serpentine configuration.